Methods of treatment using thymus-derived compositions

ABSTRACT

Embodiments of the present invention provide processes for preparing thymus extracts and plant or fungal extracts, and more particularly provide compositions (Thyex-1-6A and -6B) produced in accordance with said processes, and methods for treatment of various conditions comprising administration of said compositions including but not limited to impaired physical vigor or aptitude, and aging and/or age-related conditions (arthritis, mobility deficits, loss of appetite, etc.). Additional aspects provide methods for building muscle mass, for reducing exercise recovery period, or for sustaining exercise intensity. Particular aspects relate to preparation of  Houttuynia cordata  extracts and the use of those extracts as an anti-emetic and/or anti-nausea treatment for a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/829,829, filed on 2 Jul. 2010 and entitled METHODS OF TREATMENT USINGTHYMUS-DERIVED COMPOSITIONS (which issued on 17 Dec. 2013 as U.S. Pat.No. 8,609,824), which claims the benefit of priority to U.S. ProvisionalPatent Application Ser. Nos. 61/230,529, filed 31 Jul. 2009, entitled“METHODS OF TREATMENT USING THYMUS-DERIVED COMPOSITIONS”; 61/222,636,filed 31 Jul. 2009; entitled “METHODS OF TREATMENT OF GOUT USINGTHYMUS-DERIVED COMPOSITIONS”; and 61/228,709, filed 27 Jul. 2009,entitled “METHODS FOR TREATMENT OF CANCER USING THYMUS-DERIVEDCOMPOSITIONS”, all of which are incorporated by reference herein intheir entirety. In addition, this application is related to thefollowing co-pending patent applications: U.S. patent application Ser.No. 12/830,181, filed 2 Jul. 2010, entitled “METHODS OF TREATMENT OFGOUT USING THYMUS-DERIVED COMPOSITIONS” and International ApplicationNo. PCT/US2010/040993, filed on 2 Jul. 2010, entitled “METHODS OFTREATMENT USING THYMUS-DERIVED COMPOSITIONS”, which are incorporatedherein by reference in their entirety.

TECHNICAL FIELD OF THE INVENTION

Aspects of the present invention relate to processes for preparingthymus extracts and plant or fungal extracts, and more particularlyprovide compositions (Thyex-1-Thyex 6A and Thyex-6B) produced inaccordance with said processes, and methods comprising administration ofsaid compositions for stimulating or modulating the immune system, forbuilding muscle mass, and for treatment of various conditions includingbut not limited to impaired immune status, impaired physical vigor oraptitude, and aging and/or age-related conditions (arthritis, mobilitydeficits, loss of appetite, etc.). Combination or adjunctive therapies(e.g., with antibiotics, etc.) are also encompassed. Particular aspectsrelate to preparation of Houttuynia cordata extracts and novel uses ofsame for treating nausea (e.g., anti-nausea and/or anti-emetic).

BACKGROUND

Impaired Physical Vigor or Aptitude.

Loss of physical vigor has been associated with aging. In certaininstances, aging athletes injected with growth hormone have reported“restoration” of physical vigor. Thymic hormone has been reported toaffect the endocrine system; for example, to affect release by thepituitary of FSH and LH in thymectomized mice resulting in production oftestosterone/estrogen.

Aging.

All mammals possess a thymus gland at birth. As an animal ages, thegland begins to become fibrous and progressively degenerates. In humans,the thymus gland continues to grow until about age 20 beforedegenerating, and by age 50, no trace of glandular tissue is present.The progressive loss of the thymus can be temporally correlated to withdiminishing natural physical stamina, and increasing incidence ofage-related disorders.

There is a pronounced need in the art for economically-viable treatmentsfor immune modulation or stimulation, and the effective treatment andprevention of impaired physical vigor and stamina, and age-relateddisorders, including age-related loss of natural physical stamina.

SUMMARY OF THE INVENTION

Particular embodiments of the present invention provide inventivemethods for preparing thymus extracts (Thyex-1-6A and -6B; see workingEXAMPLES 1-8), and therapeutic compositions comprising said Thyexpreparations.

Additional exemplary embodiments provide methods for treating at leastone condition selected from the group consisting of aging and relatedconditions, restoration of athletic vigor and/or stamina (EXAMPLE 9),allergy and autoimmune disorders (e.g., lichen sclerosis set atrophicus,rheumatoid arthritis, psoriasis, progressive systematic scleroderma,lupus, and juvenile diabetes) (EXAMPLE 11), post-surgical treatmentand/or wound healing (EXAMPLE 12), emphysema, and inflammation (EXAMPLE13), comprising administration of a Thyex composition as describedherein.

In preferred embodiments, the Thyex compositions are used to stimulateor modulate the immune system, or to treat impaired physical vigor oraptitude, and aging and/or age-related conditions (arthritis, mobilitydeficits, loss of appetite, etc.).

Specifically, particular embodiments of the present invention providemethods for preparing thymus extract compositions (Thyex-1-6A and -6B;EXAMPLES 1-8) for the treatment of impaired physical vigor and stamina,and age-related disorders, comprising: homogenizing thymus tissue;removing tissue debris therefrom to produce a supernatant; andconcentrating and denaturing the supernatant to produce a clarifiedsupernatant fraction. Preferably, the processes comprise furtherclarifying of the clarified supernatant by high-speed centrifugation atabout 8,500 (g). Preferably the processes further comprise filtersterilizing. Preferably, the pH and ionic strength of the resultingsupernatant are physiologically compatible. Preferably, the pH and ionicstrength of the resulting supernatant have values of about 7 and ofabout 0.85% (w/v), respectively. Preferably, the initial ratio of thymustissue to aqueous homogenization fluid is about 350 g wet weight ofthymus tissue to about 0.7 L of homogenization fluid. Preferably, theprocesses comprise further fractionating based on molecular weight toobtain a final fraction having proteins of about 3.5 to about 30 kDa.

Additional embodiments provide processes for preparing thymus extractcompositions for the treatment of impaired physical vigor and stamina,and age-related disorders, comprising: homogenizing thymus tissue;removing tissue debris therefrom to produce a supernatant;concentrating, denaturing, and clarifying the supernatant fraction;further concentrating the clarified supernatant fraction to produce afurther concentrated fraction; fractionating the further concentratedfraction to remove molecules having a molecular weight less than about3.5 kDa; and further fractionating based on molecular weight to obtain afinal fraction having proteins of about 3.5 to about 30 kDa. Preferably,the processes further comprise adjusting the pH and/or ionic strength,of the final fraction to a physiological or therapeutically compatiblevalue. Preferably, said adjusting is achieved by adding phosphate bufferand/or sodium chloride to produce a solution having a pH value of about7, and/or an ionic strength of about 0.85% (w/v). Preferably theprocesses further comprise filter sterilizing. Preferably, saidsterilizing is achieved by using a 0.2μ membrane filter. Preferably, theinitial ratio of thymus tissue to aqueous homogenization fluid is about350 g wet weight (about 400 ml) of thymus tissue to about 0.7 L ofhomogenization fluid.

Further embodiments provide compositions for body building supplements,including protein supplements for use in building muscle mass,comprising: thymus extract compositions (Thyex-1-6A and -6B) produced inaccordance with the above-described processes, and a pharmaceuticallyacceptable carrier.

Yet further embodiments provide pharmaceutical compositions for thetreatment of impaired physical vigor and stamina, and age-relateddisorders, comprising: thymus extract compositions (Thyex-1-6A and -6B)produced in accordance with the above-described processes, and apharmaceutically acceptable carrier.

Further embodiments provide compositions for body building supplements,including protein supplements for use in building and/or increasingmuscle mass and/or as part of an exercise supplement compositioncomprising: thymus extract compositions (Thyex-1-6A and -6B) produced inaccordance with the above-described processes.

Still further embodiments provide methods for treating impaired physicalvigor and stamina, and age-related disorders, comprising: administeringof a therapeutically-effective amount to a mammal in need thereof athymus extract composition (Thyex-1-6A and -6B) produced in accordancewith the above-described processes, and wherein the mammal, includes,but is not limited to human, canine, feline, bovine, equine (e.g., racehorse), ovine, and porcine. Even further embodiments provide for methodsof treating an age-related condition wherein the age-related conditionis at least one selected from the group consisting of arthritis,mobility deficits, muscle mass loss, impaired vigor, and loss ofappetite. Even further embodiments provide for methods of treating animpaired physical vigor, stamina or aptitude, wherein the impairedphysical vigor stamina or aptitude is selected from the group consistingof decreased stamina, and impaired recovery from exercise or physicalstress. Preferably, the thymus extract composition is administered incombination with administration of macrophage stimulating agent.

According to certain aspects, the inventive Thyex compositions areuseful in reducing exercise recovery period and/or for sustainingexercise intensity, comprising: administering of atherapeutically-effective amount of a thymus extract composition(Thyex-1-6A and -6B) produced in accordance with the herein-describedprocesses.

According to additional aspects, the inventive Thyex compositions areuseful for immunostimulation and/or immunoregulation, comprisingadministering to a mammalian subject in need thereof atherapeutically-effective amount of a thymus extract composition(Thyex-1-6A and -6B) produced in accordance with the herein-describedprocesses. According to further aspects, the inventive Thyexcompositions are useful for modulating endocrine function, comprisingadministering to a mammalian subject in need thereof atherapeutically-effective amount of a thymus extract composition(Thyex-1-6A and -6B) produced in accordance with the herein-describedprocesses.

According to still further aspects, the inventive Thyex compositions areuseful for treating or preventing, virus infection, virus-associatedconditions or secondary infection, or wasting syndrome in an affected orsusceptible swine in need thereof, comprising administering to the swinea therapeutically-effective amount of a thymus extract composition(Thyex-1-6A and -6B) produced in accordance with the herein-describedprocesses.

According to certain aspects, the inventive Thyex compositions are usedin combination with administering an anti-microbial agent (e.g., anantibiotic or a Houttuynia cordata extract composition).

According to further aspects, the inventive Thyex compositions areuseful for treating nausea, comprising administration to a mammaliansubject in need thereof a therapeutically-effective amount of an extractof Houttuynia cordata.

According to additional aspects (see working EXAMPLES 14-16), theinventive Thyex compositions are used in the treatment of a disease orcondition caused by or related to the Arteriviridae family of viruses(like PRRS), PRRS-related conditions and secondary infections (e.g.,diarrhea, pneumonitis and/or intestinal disorders), and wasting syndromein pregnant gilts and sows, and in swine being fattened for slaughter.According to particular aspects, adjunct treatment of swine with Thyexcompositions is effective to enhance the efficacy of vaccine regimens inprotecting PRRSV-susceptible reproductive systems against virulent fieldstrains of PRRSV. According to further aspects, strains of PRRSV can beselected from the group consisting of strains of PRRSV of eitherEuropean or North American serotype, VR-2332 and Lelystad virus strains,NADC-8, NADC-9, and NVSL-14 strains, modified PRRSV strains, attenuatedPRRSV strains, and combinations thereof. According to yet furtheraspects, the vaccine element is a monovalent, bivalent or polyvalentPRRSV-based vaccine, or an immunogenic or antigenic component of a PRRSVstrain, or a modified or attenuated form of a PRRSV strain or PRRSVimmunogen.

Particular embodiments of the present invention provide inventivemethods for preparing palatable Houttuynia cordata extract compositionhaving anti-nausea and/or anti-emetic activity, comprising: performingan aqueous extraction of Houttuynia cordata plant material to produce aaqueous extract and an extracted plant material; separating the aqueousextract from the extracted plant material to provide a separated aqueousextract; and heat distilling a volume of the separated aqueous extractand collecting a fractional volume of initial distillate to provide asubstantially non-bitter, heat-distilled Houttuynia cordata extractcomposition having anti-nausea and/or anti-emetic activity. Furtherparticular embodiments of the present invention provide methods forpreparing palatable Houttuynia cordata extract composition, wherein atleast one of the Houttuynia cordata plant material, aqueous extract andthe separated aqueous extract is frozen.

Yet further particular embodiments of the present invention providemethods for preparing palatable Houttuynia cordata extract composition,wherein the aqueous extraction comprises aqueous extraction with heatedor boiling water. Still further particular embodiments of the presentinvention provide methods for preparing palatable Houttuynia cordataextract composition, wherein separating comprises filtering and/orcentrifugation.

According to certain aspects, the present invention provides methods forpreparing palatable Houttuynia cordata extract composition, whereinseparating comprises centrifugation, optimally at 3,500×G for 10 minutesat ambient temperature to produce a pellet, and an aqueous supernatantfraction. According to further aspects, the present invention providesmethods for preparing palatable Houttuynia cordata extract composition,wherein separating comprises centrifugation to provide an aqueoussupernatant fraction, and filtration of the aqueous supernatantfraction. According to yet further aspects, the present inventionprovides methods for preparing palatable Houttuynia cordata extractcomposition, wherein distilling comprises distilling at a temperature ofabout 100° C. or greater, and wherein distillation is allowed to proceeduntil the volume of distillate is about half of the initial primaryaqueous extract. According to still further aspects, the presentinvention provides methods for preparing palatable Houttuynia cordataextract composition, further comprising adjusting of at least one of pHand ionic strength to provide at least one of a pH-adjusted and ionicstrength-adjusted distillate fraction. According to still furtheraspects, the present invention provides methods for preparing palatableHouttuynia cordata extract composition, further comprising sterilizingof the distillate.

Particular embodiments of the present invention provide for compositionsor extracts of Houttuynia cordata plant material prepared according tothe disclosed inventive methods. Further embodiments of the presentinvention provide for methods of treating nausea, comprisingadministration to a subject in need thereof a therapeutically effectiveamount of a heat-distilled aqueous extract of Houttuynia cordata plantmaterial, or of derivative thereof having anti-nausea activity. Stillfurther embodiments of the present invention provide for methods ofscreening or identifying a composition for treating nausea, comprising:preparing a heat-distilled aqueous extract of Houttuynia cordata plantmaterial; fractionating components of the extract; and assaying at leastone fraction for anti-nausea or anti-emetic activity, or an indicatorthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagrammatic representation comprising an inventiveThyex-1 process embodiment for preparing a thymus extract composition.

FIG. 2 is a flow diagrammatic representation comprising an inventiveThyex-2 process embodiment for preparing a thymus extract composition.

FIG. 3 is a flow diagrammatic representation comprising an inventiveThyex-3 process embodiment for preparing a thymus extract composition.

FIG. 4 is a flow diagrammatic representation comprising an inventiveThyex-4, -5 and -6 process embodiments for preparing a thymus extractcomposition.

FIG. 5 is a flow diagrammatic representation comprising an inventiveD-YXC process embodiment for preparing a Houttuynia cordata extractcomposition.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the present invention relate to processes for preparingthymus extracts and plant or fungal extracts, and more particularlyprovide compositions (Thyex-1-6A and -6B) produced in accordance withsaid processes, and administration of said compositions in methods fortreatment of at least one condition selected from the group consistingof aging and related conditions, restoration of athletic vigor and/orstamina, allergy and autoimmune disorders (e.g., lichen sclerosis setatrophicus, rheumatoid arthritis, psoriasis, progressive systematicscleroderma, lupus, and juvenile diabetes), emphysema, and inflammation.

In particular aspects, administration of said compositions is used fortreatment of impaired physical vigor or aptitude, and aging and/orage-related conditions (arthritis, mobility deficits, loss of appetite,etc.).

In preferred aspects, the inventive compositions (Thyex-1-6A and -6B)are administered in combination with a macrophage stimulating agent.

Particular aspects provide a method for preparing heat-treated,fractionated thymus extract composition, comprising: homogenizing thymustissue with aqueous homogenization fluid to produce an aqueous thymushomogenate; removing tissue debris from the aqueous thymus homogenate toproduce a primary supernatant; heat denaturing the primary supernatant,and clarifying the denatured primary supernatant by use of at least oneof low-speed centrifugation and filtration, to produce a clarifiedsupernatant; and separating molecules having molecular weights less thanabout 3.5 kDa from the clarified supernatant, wherein a heat-treated,fractionated thymus extract composition lacking proteins or polypeptideshaving molecular weights less than about 3.5 kDa is provided. In certainaspects, the method further comprises separating molecules havingmolecular weights greater than about 30 kDa from the heat-treated,fractionated thymus extract composition, wherein a heat-treated,fractionated thymus extract composition comprising proteins orpolypeptides having molecular weights in the range of about 3.5 kDa toabout 30 kDa is provided. In certain embodiments, the method comprisesfurther clarifying of the clarified supernatant by high-speedcentrifugation to produce a final clarified supernatant fraction, andoptionally sterilizing the final clarified supernatant fraction toproduce a sterile final clarified supernatant fraction. In certainaspects, sterilizing is achieved by passing the final clarifiedsupernatant fraction through a membrane filter. In particularembodiments, the initial ratio of thymus tissue to aqueoushomogenization fluid is about 350 g wet weight of thymus tissue to about0.7 L of homogenization fluid. In certain aspects, removing tissuedebris from the aqueous thymus homogenate is achieved by a combinationof low-speed centrifugation and crude filtration. In certain aspects,heat denaturing and clarifying of the primary supernatant is achieved byheat denaturation, followed by low-speed centrifugation and crudefiltration to remove particulate matter. In certain embodiments, themethods further comprise lyophilization of the final clarifiedsupernatant fraction. Preferably no steps involving exogenously addedprotease digestion, or extraction with organic solvents are used.

Additional particular aspects provide a method for preparing a thymusextract composition, comprising: homogenizing thymus tissue with aqueoushomogenization fluid to produce an aqueous thymus homogenate; removingtissue debris from the aqueous thymus homogenate to produce a primarysupernatant; heat denaturing the primary supernatant, and clarifying thedenatured primary supernatant by use of at least one of low-speedcentrifugation and filtration to produce an intermediate clarifiedsupernatant; concentrating the intermediate clarified supernatant toproduce a concentrated intermediate fraction; and separating moleculeshaving molecular weights less than about 3.5 kDa from the concentratedintermediate fraction, wherein a heat-treated, fractionated thymusextract composition lacking proteins or polypeptides having molecularweights less than about 3.5 kDa is provided. In certain embodiments, themethod further comprises separating molecules having molecular weightsgreater than about 30 kDa from the heat-treated, fractionated thymusextract composition, wherein a heat-treated, fractionated thymus extractcomposition comprising proteins or polypeptides having molecular weightsin the range of about 3.5 kDa to about 30 kDa is provided. Certainembodiments further comprise clarifying of the concentrated intermediatefraction by high-speed centrifugation to produce a final clarifiedsupernatant fraction. Particular aspects further comprise adjusting atleast one of the pH or ionic strength of the fraction having proteins orpolypeptides of molecular weight of about 3.5 to about 30 kDa to aphysiological or therapeutically compatible value, to produce a pH- orionic strength-adjusted fraction, and in certain aspects, adjusting atleast one of the pH or ionic strength to a physiological ortherapeutically compatible value is achieved by adding phosphate bufferor sodium chloride to produce a fraction having at least one of a pHvalue of about 7 or an ionic strength of about 0.85% w/v. Certainembodiments further comprise sterilizing the pH-, or ionicstrength-adjusted fraction to produce a sterile pH-, or ionicstrength-adjusted fraction, and in particular aspects, sterilizing isachieved by passing the fraction through a membrane filter. Inparticular embodiments, the initial ratio of thymus tissue to aqueoushomogenization fluid is about 350 g wet weight of thymus tissue to about0.7 L of homogenization fluid. In certain aspects, removing tissuedebris from the aqueous thymus homogenate is achieved by a combinationof low-speed centrifugation and crude filtration. For particularembodiments, heat denaturing and clarifying of the primary supernatantis achieved by heat denaturation, followed by low-speed centrifugationand crude filtration to remove particulate matter. In particularembodiments, concentrating the intermediate supernatant involvesconcentrating and fractionating, wherein the concentrating andfractionating is achieved by adding ammonium sulfate to the intermediateclarified supernatant, followed by low-speed centrifugation andsuspension of the resulting ammonium sulfate pellet in an aqueoussolution to provide a concentrated intermediate fraction. In particularaspects, separating molecules having molecular weights less than about3.5 kDa from the concentrated intermediate fraction comprises dialysisof the concentrated intermediate fraction, followed by high-speedcentrifugation to remove particulate matter, to provide for a clarifiedconcentrated intermediate fraction lacking proteins or polypeptideshaving molecular weights less than about 3.5 kDa. In certainembodiments, separating molecules having molecular weights greater thanabout 30 kDa from the heat-treated, fractionated thymus extractcomposition, is achieved by passing the clarified concentratedintermediate fraction lacking proteins or polypeptides having molecularweights less than about 3.5 kDa consecutively through a first and asecond membrane filter having exclusion limits of about 100 and about 30kDa, respectively, and collecting the filtrate. Particular aspectsfurther comprise lyophilization of the heat-treated, fractionated thymusextract composition comprising proteins or polypeptides having molecularweights in the range of about 3.5 kDa to about 30 kDa.

Yet additional particular aspects provide a composition orpharmaceutical composition, comprising a thymus extract compositionproduced in accordance with the methods recited herein.

Additional aspects provide a body building supplement, comprising athymus extract composition produced in accordance with the methodsrecited herein. In certain embodiments, the supplement comprises aprotein supplement for use in building muscle mass.

Particular embodiments further comprise administering a macrophagestimulating agent in combination with administration of the thymusextract composition for use in treating at least one condition selectedfrom the group consisting of aging and related conditions, restorationof athletic vigor and/or stamina, allergy and autoimmune disorders(e.g., lichen sclerosis set atrophicus, rheumatoid arthritis, psoriasis,progressive systematic scleroderma, lupus, and juvenile diabetes),emphysema, and inflammation. In certain aspects, the macrophagestimulating agent comprises at least one of beta glucan,polysaccharides, toxoid vaccines, and Staph lysate vaccine, immunecomplexes, compliment components, lymphokinesm, tuftsin,lipopolysaccharides (LPS), muramyl dipeptide, physiologic cationcomplexing agents, pyran copolymers, polycarboxylates, ionphores,Quadrol (N,N,N′,N′-tetrakis(2-hydroxypropyl)ethlenediamine), andmacrophage stimulating peptides. In certain aspects, the beta glucancomprises beta 1,3 glucan.

Additional aspects provide a method for treating aging or an age-relatedsymptom or condition, comprising administering to a mammalian subject inneed thereof a therapeutically-effective amount of a thymus extractcomposition produced in accordance with the methods recited herein,wherein at least one age-related symptom or condition is treated oralleviated. In certain aspects the mammal is a human. In particularaspects, the age-related condition is at least one selected from thegroup consisting of arthritis, mobility deficits, muscle mass loss,impaired vigor, and loss of appetite.

Additional aspects provide a method for reducing exercise recoveryperiod or for sustaining exercise intensity, comprising administering toa mammalian subject in need thereof a therapeutically-effective amountof a thymus extract composition produced in accordance with the methodsrecited herein, wherein at least one of reducing exercise recoveryperiod, and sustaining exercise intensity is afforded. In certainaspects, the thymus extract composition is administered with, or as partof an exercise supplement composition.

Yet additional aspects provide a method for treating impaired physicalvigor or aptitude, comprising administering to a mammalian subject inneed thereof a therapeutically-effective amount of a thymus extractcomposition produced in accordance with the methods recited herein,wherein at least one symptom or condition of impaired physical vigor oraptitude is treated or alleviated. In certain aspects, the at least onesymptom or condition of impaired physical vigor or aptitude is at leastone selected from the group consisting of decreased stamina, andimpaired recovery from exercise or physical stress.

Additional aspects provide a method for increasing muscle mass,comprising administering to a mammalian subject in need thereof aneffective amount of a thymus extract composition produced in accordancewith the methods recited herein, wherein increasing muscle mass isafforded.

Certain aspects provide a method for treating arthritis and age-relatedissues, comprising administering to a mammalian subject in need thereofan effective amount of a thymus extract composition produced inaccordance with the methods recited herein, wherein effects of arthritisand age-related issues is alleviated. According to particular aspects,the mammalian subject in need of treatment includes but is not limitedto canine, feline, bovine, porcine, equine, ovine, and other largeanimals. According to further aspects, the method for treating arthritisand age-related issues includes veterinary applications. According tostill further aspects, the veterinary applications include but are notlimited to treating canine, feline, bovine, porcine, equine, ovine, andother large animals.

Further aspects provide a method for immunostimulation orimmunoregulation, comprising administering to a mammalian subject inneed thereof a therapeutically-effective amount of a thymus extractcomposition produced in accordance with the methods recited herein,wherein at least one of immunostimulation or immunoregulation isafforded.

Yet further aspects provide a method for endocrine modulation,comprising administering to a mammalian subject in need thereof atherapeutically-effective amount of a thymus extract compositionproduced in accordance with the methods recited herein, whereinendocrine modulation is afforded.

Additional aspects provide methods for treating or preventing PRRS,PRRS-associated conditions or secondary infection, and wasting syndromein an affected or susceptible swine in need thereof, comprisingadministering to the swine a therapeutically-effective amount of athymus extract composition produced in accordance with the methodsrecited herein.

DEFINITIONS

“Thymus extract” or thymus extract composition, refers to a compositionproduced in accordance with one or more of the Thyex-1, -2, -3, -4, -5,-6A and -6B processes disclosed herein.

“Houttuynia cordata” extract refers to a compositions produced inaccordance with the one or more of the D-YXC-1 and 2 processes disclosedherein.

“Animals” as used herein for treatment of subjects refers to chicken,duck, fish, hamster, rat, guinea pig, human, canine, feline, bovine,equine (e.g., race horse), ovine, goat, and porcine.

“Anti-microbial agent” means an agent with, for example, antibacterial,antifungal or antiviral activity, including, but not limited to: plantextracts (e.g., Houttuynia cordata extracts); antibiotics, such asβ-lactam antibiotics, erythromycin compounds, Tetracycline compounds,aminoglycoside antibiotics, cephalosporin compounds, anthracyclinecompounds, phleomycin group antibiotics, sulfonamide compounds,macrolide antibiotics (e.g., tylosin, desmycosin, macrocin, andlactenocin), quinolone and quinolonyl compounds (e.g., quinolonyllactams and quinolone thioureas, and carbacephem- andcarbapenem-quinolones) carbapenem compounds, along with those antibioticagents more commonly used in the swine industry, such aslankacidin-group antibiotics and derivatives, diterpene antibiotics(e.g., tiamulin-type), polyether or polycyclic ether antibiotics andderivatives (e.g., A82810), lysocellin, treponemycin, antibiotic 10381b,antibiotics GE 37468 A, B and C, A41030 antibiotics, antibiotic A47934,antibiotic BN-109, apramycin, actaplanin antibiotics, antibiotic A3823,antibiotic X-14766A, dihydromocimycin antibiotics, BM123γ-typeantibiotics, antibiotic AV290, antibiotic A-32887, glycopeptideantibiotic UK-68,597, valnemulin, tiamulin, oxytetracyclin,chlortetracycline, tylosin, and manganese-containing antibiotic agents,copper-containing bleomycin group antibiotics; antifungal agents, suchas partanamicins, fusacandins; and antihelminthic agents such asspiroketals, avermectin and milbemycin; and combinations thereof.

“Crude filtration” or “coarse filtration” means filtering a solutionhaving particulate, precipitated or flocculent suspended materialthrough, e.g., one or more layers of standard cheese cloth, or othersieving device (e.g., screen, strainer, colander, etc.), to remove saidmaterial.

“Low-speed centrifugation” means centrifugation at about 3,500×g (±5% or±10%) for about 5-10 minutes (±5% or ±10%), or an equivalentsedimentation protocol thereof.

“High-speed centrifugation” means centrifugation at about 8,500×g (±5%or ±10%) for about 10 minutes (±5% or ±10%), or the equivalentsedimentation protocol thereof.

“Clarifying,” or clarification of a supernatant fraction means removingparticulate matter (e.g., precipitates, bacteria) from a solutioncontaining such particulate matter through the use of standardseparation techniques, such as low- or high-speed centrifugation (asdefined above) or filtration.

With respect to fractionation of the particular supernatant fractions,the phrase “less than about 3.5 kDa” as used herein refers to less than3.5 kDa, or less than a molecular weight that varies by ±5% or ±10%therefrom. Similarly, the phrase “proteins or polypeptides of molecularweight of about 3.5 to about 30 kDa” as used herein refers to proteinsor polypeptides in a molecular weight ranged from 3.5 kDa, or from amolecular weight that varies by ±5% or ±10% therefrom, to 30 kDa, or toa molecular weight that varies by ±5% or ±10% therefrom.

With respect to pH and ionic strength, the phrase “a pH value of about7, or an ionic strength of about 0.85% w/v.” as used herein refers to apH of 7 or a pH that varies by ±5% or ±10% therefrom, and/or an ionicstrength of 0.85% w/v, or an ionic strength that varies by ±5% or ±10%therefrom.

“Vaccine” is defined herein in its broad sense to refer to any type ofbiological agent, administrable for the purpose of priming, enabling orenhancing an immune response against in an animal inoculated with thevaccine.

“Unpalatable,” as used herein, refers to the art-recognized off-puttingand/or bitter flavor widely recognized in the context of Houttuyniacordata extract. For example, those familiar with Houttuynia cordataextract (e.g., tea) described it as being bitter and/or fishy and thatthis flavor renders the extract largely unpalatable. The disclosedinvention not only provides for separation of the unpalatable andpalatable portions but also allows for separation of the anti-nauseaand/or anti-emetic activity from the largely unpalatable portion usingthe heat-distilled technique as herein disclosed. The term “separation,”as used herein can mean either separation of the unpalatable taste fromthe palatable taste in particular embodiments, or in alternateembodiments can mean loss of the unpalatable taste. “Unpalatable”, asused herein, refers in particular embodiments to the non-heat-distilledHouttuynia cordata extract (e.g., the aqueous extract and the separatedaqueous extract) being unpleasant, inedible, indigestible, disgusting,revolting, foul-tasting, nasty, bad, distasteful, disagreeable, bitter,offensive, unattractive, horrid, unsavory, displeasing, and repugnant.

“Bitter” and “bitterness,” as used herein, refers in particularembodiments to the flavor the non-heat-distilled Houttuynia cordataextract (e.g., the aqueous extract and the separated aqueous extract).In particular, bitter refers to being or inducing the one of the fourbasic taste sensations that is particularly acrid, astringent, ordisagreeable and suggestive of an infusion of hops.

“Substantially non-bitter,” as used herein, refers in particularembodiments to reducing the bitterness/foulness of the extract byapproximately half of the original non-heat-distilled Houttuynia cordataextract (e.g., the aqueous extract and the separated aqueous extract) orby reducing the bitterness/foulness to such a level that one wouldreasonably regard the solution as being palatable and/or without aunpleasant, inedible, indigestible, disgusting, revolting, foul-tasting,nasty, bad, distasteful, disagreeable, bitter, offensive, unattractive,horrid, unsavory, displeasing, and repugnant flavor. “Substantiallynon-bitter,” as used herein, refers in particular embodiments toreducing the bitterness/foulness of the extract by approximately 60%.Preferably, particular embodiments relate to reducing thebitterness/foulness of the extract by approximately 70%. Morepreferably, particular embodiments relate to reducing thebitterness/foulness of the extract by approximately 75%. Even morepreferably, particular embodiments relate to reducing thebitterness/foulness of the extract by approximately 80%. Still morepreferably, particular embodiments relate to reducing thebitterness/foulness of the extract by approximately 85%. Mostpreferably, particular embodiments relate to reducing thebitterness/foulness of the extract by approximately 90%.

Methods for Preparing Thymus Extracts:

Particular embodiments of the present invention (see working EXAMPLES1-8) provide novel processes for preparing therapeutically usefulextracts (Thyex-1-6A and -6B) of thymus tissue. In particular aspects,the inventive processes are readily distinguishable from other knownprocesses for preparing thymus extracts (e.g., Goldstein & White,Contemp. Topics in Immunobiology, p339, 1973; Bergesi et al., FoliaAllergol. Immunol. Clin. 21:201, 1977; Hooper et al., “The purificationand properties of bovine thymosin,” Ann. NY Acad. Sci. 249:125, 1975;U.S. Pat. No. 4,826,680, issued 2 May 1989 to Jaeger, PharmaceuticalComposition Containing Thymus Extract Fractions), and lack stepsinvolving decalcite (CaCO₃) treatment, protease digestion, extractionwith organic solvents (e.g., phenol, acetone or ethanol) orfractionation by column chromatography. Not only are the inventivecompositions surprisingly effective in view of the teachings of the art,but the compositions produced in accordance with the instant processesare also further distinguished from those of the prior art by themolecular weight ranges of their protein elements.

The instant processes comprise steps to optimize protein compositionsfor therapeutic use of. For example, particular of the below-describedprocess embodiments (Thyex-1-6A and -6B) are designed to providetherapeutic compositions, and include ammonium sulfateprecipitation/fractionation and/or lyophilization steps, respectively,to facilitate optimal protein concentration and fractionation. TheThyex-3 process embodiment lacks an ammonium sulfate or lyophilizationstep, but provides for a sufficiently-concentrated composition byreusing (and thereby augmenting) an initial tissue homogenizationsupernatant fraction as homogenization fluid to homogenize additionaltissue. The resulting Thyex-3 composition is less refined relative tothose of Thyex-1 and Thyex-2, but is nonetheless suitably concentratedand formulated for efficacious delivery. The Thyex 6A and Thyex 6Bprocess embodiments described below are designed to provide therapeuticcompositions suitable for delivery as a topical ointment or by injectionor inhalation, and include ammonium sulfate precipitation/fractionationsteps. Thyex 5 is prepared from a similar process but is less refined(less fractionated) than Thyex 6A or Thyex 6B and is optimally mixedwith an amount of an extracted lyophilized herbal source composition,and administered orally in filled gelatin capsules. The Thyex 4 processembodiment lacks ammonium sulfate precipitation step but compriseslyophilization to provide for a sufficiently-concentrated composition.The resulting Thyex 4 composition is less refined in relative to thoseof Thyex 5 or Thyex 6A or 6B, but is nonetheless suitably concentratedand formulated for efficacious oral deliver in both animals and humans.

Preferably, the thymus preparations are those comprising Thyex-4,5-, -6Aand -6B (see FIG. 4, and EXAMPLES 4-8).

Particular specific aspects provide a method for preparing a thymusextract composition, comprising: homogenizing thymus tissue with aqueoushomogenization fluid to produce an aqueous thymus homogenate; removingtissue debris from the aqueous thymus homogenate to produce a primarysupernatant; and heat denaturing and clarifying the primary supernatantto produce a clarified supernatant. In certain aspects, the methodfurther comprises further clarifying of the clarified supernatant byhigh-speed centrifugation to produce a final clarified supernatantfraction. In certain embodiments, the method further comprisessterilizing the final clarified supernatant fraction to produce asterile final clarified supernatant fraction. In particular aspects,sterilizing is achieved by passing the final clarified supernatantfraction through a membrane filter. In particular implementations, theinitial ratio of thymus tissue to aqueous homogenization fluid is about350 g wet weight of thymus tissue to about 0.7 L of homogenizationfluid. In certain aspects, removing tissue debris from the aqueousthymus homogenate is achieved by a combination of low-speedcentrifugation and crude filtration. In particular aspects, heatdenaturing and clarifying of the primary supernatant is achieved by heatdenaturation, followed by low-speed centrifugation and crude filtrationto remove particulate matter. In certain implementations, the methodfurther comprises lyophilization of the final clarified supernatantfraction.

Additional aspects provide a method for preparing a thymus extractcomposition, comprising: homogenizing thymus tissue with aqueoushomogenization fluid to produce an aqueous thymus homogenate; removingtissue debris from the aqueous thymus homogenate to produce a primarysupernatant; heat denaturing and clarifying the primary supernatant toproduce an intermediate supernatant; and concentrating the intermediatesupernatant to produce a concentrated intermediate fraction. In certainaspects, the method further comprises further clarifying of theconcentrated intermediate fraction by high-speed centrifugation toproduce a final clarified supernatant fraction. In particularembodiments, the method further comprises fractionating the finalclarified supernatant fraction to remove molecules having a molecularweight less than about 3.5 kDa to produce a fractionated intermediatefraction. In certain aspects, the method further comprises fractionatingthe fractionated intermediate fraction, based on molecular weight, toobtain a fraction having proteins of about 3.5 to about 30 kDa. Inparticular implementations, the method further comprises adjusting atleast one of the pH or ionic strength of the fraction having proteins ofabout 3.5 to about 30 kDa to a physiological or therapeuticallycompatible value, to produce a pH- or ionic strength-adjusted fraction.In certain aspects, adjusting at least one of the pH or ionic strengthto a physiological or therapeutically compatible value is achieved byadding phosphate buffer or sodium chloride to produce a fraction havingat least one of a pH value of about 7 or an ionic strength of about0.85% w/v. In certain aspects, the method further comprises sterilizingthe pH-, or ionic strength-adjusted fraction to produce a sterile pH-,or ionic strength-adjusted fraction. In particular embodiments,sterilizing is achieved by passing the fraction through a membranefilter. In certain aspects, the initial ratio of thymus tissue toaqueous homogenization fluid is about 350 g wet weight of thymus tissueto about 0.7 L of homogenization fluid. In particular embodiments,removing tissue debris from the aqueous thymus homogenate is achieved bya combination of low-speed centrifugation and crude filtration. Incertain aspects, heat denaturing and clarifying of the secondarysupernatant is achieved by heat denaturation, followed by low-speedcentrifugation and crude filtration to remove particulate matter. Inparticular implementations, concentrating the intermediate supernatantinvolves concentrating and fractionating, and wherein the concentratingand fractionating is achieved by adding ammonium sulfate to theintermediate supernatant, followed by low-speed centrifugation andsuspension of the resulting ammonium sulfate pellet in an aqueoussolution. In some embodiments, fractionating the concentratedintermediate fraction to remove molecules having a molecular weight lessthan about 3.5 kDa is achieved by dialysis of the concentratedintermediate fraction, followed by high-speed centrifugation to removeparticulate matter. In particular aspects, fractionating thefractionated intermediate fraction, based on molecular weight, isachieved by passing the fractionated intermediate fraction consecutivelythrough a first and a second membrane filter having exclusion limits ofabout 100 and about 30 kDa, respectively, and collecting the filtrate.In certain aspects, the method further comprises lyophilization of thefraction having proteins of about 3.5 to about 30 kDa.

Particular specific aspects provide a process for preparing a thymusextract composition, comprising: homogenizing thymus tissue with aqueoushomogenization fluid to produce an aqueous thymus homogenate; removingtissue debris from the aqueous thymus homogenate to produce a primarysupernatant; concentrating the primary supernatant to produce asecondary supernatant; and denaturing and clarifying the secondarysupernatant to produce a clarified supernatant. In certain embodiments,the method further comprises further clarifying of the clarifiedsupernatant by high-speed centrifugation to produce a final clarifiedsupernatant fraction. In particular embodiments, the method furthercomprises sterilizing the final clarified supernatant fraction toproduce a sterile final clarified supernatant fraction. In certainimplementations, sterilizing is achieved by passing the final clarifiedsupernatant fraction through a membrane filter. In certain aspects, theinitial ratio of thymus tissue to aqueous homogenization fluid is about300 g wet weight, or about 340 ml wet volume, of thymus tissue to about0.8 L of homogenization fluid. In certain aspects, removing tissuedebris from the aqueous thymus homogenate is achieved by a combinationof low-speed centrifugation and crude filtration. In particularembodiments, concentrating the primary supernatant is achieved byrepeating (a) and (b) using the primary supernatant, in place of theaqueous homogenization fluid, for homogenizing additional thymus tissue.In certain aspects, denaturing and clarifying of the secondarysupernatant is achieved by heat denaturation, followed by low-speedcentrifugation and crude filtration to remove particulate matter.

Additional specific aspects provide a method for preparing a thymusextract composition, comprising: homogenizing thymus tissue with aqueoushomogenization fluid to produce an aqueous thymus homogenate; removingtissue debris from the aqueous thymus homogenate to produce a primarysupernatant; concentrating the primary supernatant to produce asecondary supernatant; denaturing and clarifying the secondarysupernatant to produce an intermediate supernatant; concentrating theintermediate supernatant to produce a concentrated intermediatefraction; fractionating the concentrated intermediate fraction to removemolecules having a molecular weight less than about 3.5 kDa to produce afractionated intermediate fraction; and fractionating the fractionatedintermediate fraction, based on molecular weight, to obtain a fractionhaving proteins of about 3.5 to about 30 kDa. In certain embodiments,the method further comprises adjusting at least one of the pH or ionicstrength of the fraction having proteins of about 3.5 to about 30 kDa toa physiological or therapeutically compatible value, to produce a pH- orionic strength-adjusted fraction. In particular implementations,adjusting at least one of the pH or ionic strength to a physiological ortherapeutically compatible value is achieved by adding phosphate bufferor sodium chloride to produce a fraction having at least one of a pHvalue of about 7 or an ionic strength of about 0.85% w/v. In someaspects, the method further comprises sterilizing the pH-, or ionicstrength-adjusted fraction to produce a sterile pH-, or ionicstrength-adjusted fraction. In particular embodiments, sterilizing isachieved by passing the fraction through a membrane filter. In certainaspects, the initial ratio of thymus tissue to aqueous homogenizationfluid is about 350 g wet weight of thymus tissue to about 0.7 L ofhomogenization fluid. In certain embodiments, removing tissue debrisfrom the aqueous thymus homogenate is achieved by a combination oflow-speed centrifugation and crude filtration. In certain aspects,concentrating the primary supernatant is achieved by repeating (a) and(b) using the primary supernatant, in place of the aqueoushomogenization fluid, for homogenizing additional thymus tissue. Inparticular implementations, denaturing and clarifying of the secondarysupernatant is achieved by heat denaturation, followed by low-speedcentrifugation and crude filtration to remove particulate matter. Inparticular aspects, the intermediate supernatant is concentrated,wherein concentrating is achieved by lyophilizing the intermediatesupernatant either to complete dryness followed by aqueous resuspensionto about 500 ml/13.6 kg (30 lbs.) original wet tissue, or to a volume ofabout 10% of its original volume. In particular aspects, concentratingthe intermediate supernatant involves concentrating and fractionating,and wherein the concentrating and fractionating is achieved by addingammonium sulfate to the intermediate supernatant, followed by low-speedcentrifugation and suspension of the resulting ammonium sulfate pelletin an aqueous solution. In certain embodiments, fractionating theconcentrated intermediate fraction to remove molecules having amolecular weight less than about 3.5 kDa is achieved by dialysis of theconcentrated intermediate fraction, followed by high-speedcentrifugation to remove particulate matter. In particular aspects,fractionating the fractionated intermediate fraction, based on molecularweight, is achieved by passing the fractionated intermediate fractionconsecutively through a first and a second membrane filter havingexclusion limits of about 100 and about 30 kDa, respectively, andcollecting the filtrate.

Additional aspects provide a pharmaceutical composition, comprising athymus extract composition produced in accordance with one or more ofthe processes disclosed herein.

Methods of Treating:

The term “treating” refers to, and includes, reversing, alleviating,inhibiting the progress of, or preventing a disease, disorder orcondition, or one or more symptoms thereof; and “treatment” and“therapeutically” refer to the act of treating, as defined herein.

A “therapeutically-effective amount” is any amount of any of thecompounds utilized in the course of practicing the invention providedherein that is sufficient to reverse, alleviate, inhibit the progressof, or prevent a disease, disorder or condition, or one or more symptomsthereof.

According to particular aspects the methods comprise administration of acomposition comprising at least one of Thyex-1-6A and -6B, as definedherein, in combination with (e.g., adjunctive therapy) administration ofa macrophage stimulating agent.

According to particular aspects, a polysaccharide is used as preferredmacrophage stimulating agent. In preferred aspects, the macrophagestimulating agent comprises a beta glucan. In particular embodiments,the beta glucan comprises at least one linkage selected from the groupconsisting of beta: 1,3; 1,4; and 1,6 glucan linkages. Preferably, thelinkage is that of beta 1,3 glucan.

According to particular aspects the inventive Thyex compositions areused in adjunctive therapies with extracts of at least one of: Paresiscrepe (aka cauliflower mushroom or hanabaritake) preparations comprisingbeta 1-3 glucan; Lentinula edodes (shitake; e.g., alkaline digestaccording to the procedure reported by Ohno et al. (Biol. Phar. Bull. 23866-872, 2000), comprises beta 1-3 glucan and chitin; Astralagasmembranaceus; Scutellaria baicalensis; Lilium longiforum (aka Easterlily); and Houttuynia cordata extracts.

Additional aspects provide a pharmaceutical composition, comprising athymus extract composition produced in accordance with one or more ofthe processes disclosed herein.

Combination therapies. Combination therapies are also encompassed byaspects of the present invention. For example, the inventive methods mayfurther comprise administration of a therapeutically-effective amount ofone or more anti-microbial agents, such as anti-viral agents,anti-bacterial agents, and anti-fungal agents. Examples of anti-viralagents include but are not limited to: combivir, boceprevir, abacavir,docosanol, aciclovir, didanosine, cidofovir, acyclovir, delavirdine,adefovir, amantadine, amprenavir, arbidol, darunavir atazanavir,atripla, zanamivir, and oseltamivir. Examples of anti-bacterial agentsinclude but are not limited to: metronidazole, tinidazole,co-trimoxazole, cephamandole, ketoconazole, latamoxef, cefoperazone,amoxicillin, cefmenoxime, furazolidone, doxycycline and erythromycin.Examples of anti-bacterial agents include but are not limited to:imidazoles, (eg., miconazole, ketoconazole, clotrimazole, econazole,bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole,sertaconazole, sulconazole, and tioconazole), triazoles (e.g.,fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole,voriconazole, and terconazole), thiazoles (e.g., abafungin), allylamines(e.g., terbinafine, amorolfine, naftifine, and butenafine), andechinocandins (e.g., anidulafungin, caspofungin, and micafungin).

Methods for Preparation of Houttuynia cordata Extracts:

Additional embodiments of the present invention (see working EXAMPLE 10)provide methods for preparing therapeutic extracts (D-YXC-1 and D-YXC-2)from the medicinal herb Houttuynia cordata Thunb. The processes compriseaqueous extraction and distillation steps.

Methods for Treatment of Aging and Related Conditions, and Restorationof Athletic Vigor and Stamina:

According to additional aspects (see working EXAMPLE 9), the endocrinesystem is also involved in the aging process, and the inventive Thyexcompositions have substantial utility for additionally affecting aspectsof the endocrine system, and have utility for treatment of aging andrelated conditions, and restoration of athletic vigor and stamina.

Without being bound by mechanism, these observations are explained, atleast in part, by implicating pituitary release of growth hormone. Itshould be noted that levels of growth hormone in pituitary remainsconstant regardless of age.

According to particular aspects, Thyex directs the hypothalamus toresume release of growth-hormone-releasing hormone, which apparentlydecreases as animal ages.

Therefore, as an athlete ages there is a loss of vigor and/or stamina.According to particular aspects, and without being bound by theory,Thyex treatment benefits athletes, and particularly athletes in their30's and older, who can regain lost stamina, and improved recovery fromstressful exercises, etc.

Yet further specific exemplary aspects, provide a method for treatingaging or age-related conditions, comprising administering to a subjectin need thereof a therapeutically-effective amount of a thymus extractcomposition produced in accordance with one or more of the processesdisclosed herein. In certain embodiments, the age-related condition isat least one selected from the group consisting of arthritis, mobilitydeficits, impaired vigor, and loss of appetite.

Additional aspects provide a method for treating impaired physical vigoror aptitude, comprising administering to a subject in need thereof atherapeutically-effective amount of a thymus extract compositionproduced in accordance with one or more of the processes disclosedherein. In certain aspects, treating impaired physical vigor or aptitudeis treatment of at least one selected from the group consisting of loststamina, and impaired recovery from physical stress.

Preferred embodiments relates to a method for treating arthritis andage-related issues, comprising administering to a mammalian subject inneed thereof an effective amount of a thymus extract compositionproduced in accordance with the methods recited herein, wherein effectsof arthritis and age-related issues is alleviated. In further preferredembodiments, the mammalian subject in need of treatment includes but isnot limited to canine, feline, bovine, porcine, equine, ovine, and otherlarge animals. In yet further preferred embodiments, the method fortreating arthritis and age-related issues includes veterinaryapplications. In additional preferred embodiments, the veterinaryapplications include but are not limited to treating canine, feline,bovine, porcine, equine, ovine, and other large animals.

Methods for Treatment of Allergy and/or Autoimmune Disease:

According to additional aspects (see working EXAMPLE 11), the inventiveThyex compositions are used to treat individuals with allergy andautoimmune disorders (lichen sclerosis set atrophicus, rheumatoidarthritis, psoriasis, progressive systematic scleroderma, lupus, andjuvenile diabetes).

Without being bound by theory, the mechanism likely comprisesstimulation of suppressor T cells, which direct B cells producing theallergy antibodies to stop continued activity and control of reactive Tcells.

Methods for Post-Surgical Treatment and/or Wound Healing:

According to additional aspects (see working EXAMPLE 12), the inventiveThyex compositions are used in post-surgical treatment, and/or forimproved wound healing.

Methods for Treatment of Emphysema:

According to additional aspects (see working EXAMPLE 13), the inventiveThyex compositions are used in the Treatment of emphysema.

Methods for Treatment of PRRS, PRRS-Related Conditions and SecondaryInfections:

According to additional aspects (see working EXAMPLES 15-17), theinventive Thyex compositions are used in the Treatment of PRRS,PRRS-related conditions and secondary infections (e.g., diarrhea,pneumonitis and/or intestinal disorders), and wasting syndrome inpregnant gilts and sows, and in swine being fattened for slaughter.According to particular aspects, adjunct treatment of swine with Thyexcompositions is effective to enhance the efficacy of vaccine regimens inprotecting PRRSV-susceptible reproductive systems against virulent fieldstrains of PRRSV.

According to particular aspects, adjunct treatment of swine with Thyexcompositions is effective to enhance protective immunity to PRRSVinfection, even during late gestation.

According to further aspects, adjunctive treatment of swine with Thyexcompositions is effective to enhance the efficacy of vaccine regimens inprotecting PRRSV-susceptible respiratory systems against PRRSV andPRRSV-related conditions.

According to yet further aspects, adjunctive treatment of swine withThyex compositions is effective to reducing vaccine-induced reproductivefailure when such vaccines are administered during gestation (includinglate gestation), allowing for the use of less attenuated and/or morebroadly protective vaccines.

Additional aspects provide a method for treating or preventing PRRS,PRRS-associated conditions or secondary infection, and wasting syndromein an affected or susceptible swine in need thereof, comprisingadministering to the swine a therapeutically-effective amount of athymus extract composition produced in accordance with one or more ofthe processes disclosed herein. In certain aspects the method furthercomprises administering an anti-microbial agent in combination withadministration of the thymus extract composition. In particularimplementations, the anti-microbial agent is an antibiotic or aHouttuynia cordata extract composition.

Further aspects, provide a method for treating or preventing, virusinfection, virus-associated conditions or secondary infection, orwasting syndrome in an affected or susceptible swine in need thereof,comprising administering to the swine a therapeutically-effective amountof a thymus extract composition produced in accordance with one or moreof the processes disclosed herein as an adjunct treatment in combinationwith administration of a vaccine element or regimen. In certain aspectsthe method further comprises administering an anti-microbial agent incombination with administration of the thymus extract composition. Inparticular implementations, the anti-microbial agent is an antibiotic ora Houttuynia cordata extract composition. In particular aspects, thevaccine element or regimen is based on one or more strains ofArteriviridae family viruses (e.g., comprises a PRRSV strain, whereinthe PRRSV strain is selected from the group consisting of strains ofPRRSV of either European and North American serotype, VR-2332 andLelystad virus strains, NADC-8, NADC-9, and NVSL-14 strains, modifiedPRRSV strains, attenuated PRRSV strains, and combinations thereof). Incertain aspects, the vaccine element is a monovalent, bivalent orpolyvalent PRRSV-based vaccine, or an immunogenic or antigenic componentof a PRRSV strain, or a modified or attenuated form of a PRRSV strain orPRRSV immunogen.

Yet additional aspects provide a method for treating or preventingvaccine-induced reproductive failure in a swine in need thereof,comprising administering to the swine a therapeutically-effective amountof a thymus extract composition produced in accordance with one or moreof the processes disclosed herein as an adjunct treatment in combinationwith administration of a vaccine element or regimen. In certainembodiments, the method further comprises administering ananti-microbial agent in combination with administration of the thymusextract composition.

Methods for Treatment of Nausea and/or Vomiting Using Heat-DistilledHouttuynia cordata Extract.

According to additional aspects (see working EXAMPLES 10 and 17-20), theinventive heat-distilled Houttuynia cordata extract is useful in thetreatment of nausea and/or vomiting in an affected or susceptiblesubject in need thereof, comprising administering to the subject atherapeutically-effective amount of a heat-distilled Houttuynia cordataextract composition produced in accordance with one or more of theprocesses disclosed herein. In certain aspects, the method furthercomprises administering an additional anti-nausea and/or anti-emeticagent in combination with administration of the heat-distilledHouttuynia cordata extract composition. In further aspects, the methodcomprises administering an additional anti-nausea and/or anti-emeticagent sequentially with administration of the heat-distilled Houttuyniacordata extract composition. In yet further aspects, the methodcomprises administering an additional anti-nausea and/or anti-emeticagent at a similar time with administration of the heat-distilledHouttuynia cordata extract composition.

According to additional aspects (see working EXAMPLES 10 and 17-20), theinventive heat-distilled Houttuynia cordata extract is useful in thetreatment of nausea and/or vomiting brought on by any conditionincluding, but not limited to pregnancy (e.g., morning sickness), motionsickness, gastrointestinal obstruction, peptic ulcer, drug toxicity,myocardial infarction, renal failure, and hepatitis. In addition, nauseaand/or vomiting can be the side effect of many cancer treatments,including but not limited to chemotherapeutic drugs, radiation, andsurgery.

According to further aspects, the inventive heat-distilled Houttuyniacordata extract has substantial utility to treat nausea in both humansand animals caused by illnesses, infections, or other treatments, and inparticular embodiments is used in combination with one or more of theinventive Thyex compositions, plus or minus standard chemotherapy drugs,for the treatment of cancer.

In further aspects the method further comprises administering theheat-distilled Houttuynia cordata extract composition with a standardchemotherapy drugs, for the treatment of cancer. Standardchemotherapeutic drugs are well known in the art. In yet further aspectsthe method comprises administering the heat-distilled Houttuynia cordataextract composition in combination with a chemotherapeutic agent,wherein the administration can occur simultaneously, sequentially, or asneeded to relieve nausea and vomiting symptoms.

Compositions:

Additional embodiments provide compositions produced in accordance withsaid processes. The Thyex-1, -2, -3, -4, -5, -6A and -6B compositionembodiments are produced in accordance with the corresponding Thyex-1-6Aand -6B processes (Working EXAMPLES 1-8). Likewise, the inventiveheat-distilled Houttuynia cordata extract (D-YXC-1 and 2) compositionembodiments are produced in accordance with the corresponding D-YXC-1,and 2 processes (EXAMPLE 10).

Methods of Treatment:

Further embodiments provide methods for the treatment of impairedphysical vigor and stamina, and age-related disorders, comprising:utilizing thymus extract compositions alone, or in combination with amacrophage stimulating agent (see working EXAMPLE 9 below).

Treatment in Humans with Thyex-1-6A and 6B Compositions, with andwithout Macrophage Stimulating Agents.

Thyex-1-3 Processes.

EXAMPLES 1-3 provide exemplary process embodiments used for preparingThyex-1-3, produced in accordance therewith suitable for oral delivery.Alternatively, Thyex-1-3 are lyophilized, stored at ambient temperatureand reconstituted with sterile water prior to use.

Thyex-4 Process.

Steps (1)-(11) of EXAMPLE 4 comprise a process embodiment for producingThyex-4 (step (12) relates to storage), suitable for oral delivery.Alternatively, Thyex-4 is lyophilized, stored at ambient temperature andreconstituted with sterile water prior to use.

Thyex-5 Process.

Steps (1)-(13) EXAMPLE 5 comprise a process embodiment for producingThyex-5 (step (14) relates to storage), suitable for oral delivery.Alternatively, Thyex-5 is lyophilized, stored at ambient temperature andreconstituted with sterile water prior to use.

Thyex-6A Process.

EXAMPLE 6 provides an exemplary process embodiment used for preparingThyex-6A produced in accordance therewith suitable for oral delivery, ordelivery as a topical ointment or by injection or inhalation.Alternatively, Thyex-6A is lyophilized, stored at ambient temperatureand reconstituted with sterile water prior to use.

Thyex-6B Process.

EXAMPLE 7 provides an exemplary process embodiment used for preparingThyex-6B produced in accordance therewith suitable for oral delivery, ordelivery as a topical ointment or by injection or inhalation.Alternatively, Thyex-6B was lyophilized, stored at ambient temperatureand reconstituted with sterile water prior to use.

Treatment in Swine with Thyex-1-3 Compositions, with and withoutAntimicrobial Agents.

In Swine, the treatment methods utilizing the Thyex-1, -2 and -3composition embodiments comprise for example, intra-muscular orsubcutaneous injections thereof into affected swine to treat the primaryPRRSV infection. Reproductive and maturational deficits, includingearly-stage PRRS symptoms (e.g., lethargy, anorexia, elevated bodytemperatures of 103-104° F.), were effectively treated with theadministration of Thyex-1 or 2 alone. Preferably, PRRSV-infected animalsare treated with a either a three-day, or up to about a 7-day regimenconsisting of daily injections (delivered either intramuscularly (IM) orsubcutaneously (SQ)) of 1 ml Thyex-1 or 2 composition.

Thyex-1 and 2 treatments were supplemented with administration ofantimicrobial agents (e.g., either antibiotics or D-YXC-1 or 2) foradvanced PRRS stages characterized by local or systemic secondaryinfections, such as pneumococcus and salmonella. In these instances, theantimicrobial agent was generally co-administered (by injection) witheither the Thyex-1 or 2 composition. For example, for Thyex-1 or 2 plusD-YXC-1 combination therapy, PRRSV-infected animals were treated with aeither a three-day, or up to about a 7-day regimen consisting of dailyinjections (delivered either intramuscularly (IM) or subcutaneously(SQ)) of 1 ml Thyex-1 or 2 composition, in combination withadministration of the antimicrobial agent (e.g., either antibiotics orD-YXC-1 or 2). Alternatively, combination dosage regimes that involved aone-day course of two or three 1 ml injections of Thyex-1 or 2 over thecourse of a single day, combined with a two- or three-day course ofantimicrobial agent administration were found to be therapeuticallyeffective.

The antimicrobial agent (e.g., antibiotic or D-YXC-2 extract)composition was co-administered, by oral delivery, with theabove-described Thyex-1 or 2 dosage regimens when the late-stagesecondary infection was gastrointestinal. For example, D-YXC-2 (5 ml forsucklings or weanlings, and up to 15 ml for adult swine) was orallyadministered three times daily for two or three days, in conjunctionwith Thyex-1 or 2 injections.

Treatment with Thyex-3.

The treatment methods utilizing the Thyex-3 composition embodimentcomprise oral delivery thereof into affected swine to treat the primaryPRRSV infection. Early stages of PRRS, as was found with the Thyex-1 and2 compositions, were effectively treated with the administration of theThyex-3 composition alone. In this instance, about 15 ml of the Thyex-3composition was delivered daily for 30 consecutive days. Preferably, theThyex-3 compositions are standardized to a protein concentration of 2mg/ml.

Thyex-3 treatment was supplemented with antimicrobial agents (e.g.,antibiotics; or D-YXC-1 or 2 extracts, for pneumonitis/systemic vs.gastrointestinal, respectively) for advanced PRRS stages in the samemanner and dosage regimens as described above for co-administration withthe Thyex-1 and 2 treatments.

Compositions produced in accordance with the processes of the presentinvention can be administered to breeding gilts, sows, boars, sucklingsor weaned piglets. Preferably, the compositions are administered tobreeding females before mating, or to pregnant animals to help protectthe entire pregnancy period. Preferably, the compositions areadministered to young sucklings and weanlings to protect their latenursery, grower and finishing stages.

Treatment of Thyex-1, 2, or 3 in Combination with Vaccines forPreventing PRRSV Infection.

The inventive compositions are useful in adjunct therapies, and for thispurpose are administered in relation to administration of PRRSV vaccineelements or vaccination regimens, to enhance vaccine efficacy. Suchvaccines or vaccine elements include any administrable agent that iscapable of promoting or enhancing a PRRSV-protective immune response inanimals inoculated with the vaccine or vaccine element. PRRSV vaccinesor vaccine elements are well known in the art (see “Background,” hereinabove) and include, but are not limited to attenuated, “modified-live”or inactivated viruses or viral elements, viral antigens, monovalent(based on a single PRRSV strain), bivalent or even polyvalent vaccines(based on 2 or 3 strains; see, e.g., U.S. Pat. No. 5,976,537), variousadjuvants, and combinations thereof, and may correspond to one or moregenetically diverse field strains of PRRSV.

Adjunct therapy comprises administration of the inventive Thyex, and/orThyex plus antimicrobial agent (e.g., antibiotic, or Houttuynia cordataextract) to swine at or about the time of initial vaccination and/or ofsubsequent vaccine boosts.

Therapeutic Utility of Thymus Extracts for the Treatment of PRRS.

The treatment methods involving administration of Thyex-1, 2 or 3, aloneor in combination with antimicrobial agents, were effective for pregnantgilts and sows, and for swine being fattened for slaughter (see Example3, below). Post-farrowing gilts or sows showed a more rapid return toestrus and a higher pregnancy rate. Moreover, the treated animalsrecovered more rapidly, relative to non-treated control animals, fromPRRS-associated secondary infections, such as pneumonia or salmonella.

Treated PRRSV-infected animals, in contrast to non-treated PRRS-Vinfected animals, had healthy appetites and reached full maturity withinthe same time period “on feed” as control animals that were not infectedwith PRRSV.

According to the present invention, adjunctive treatment of swine withThyex (or Thyex plus antimicrobial agent) compositions enhancesprotective immunity to PRRSV, even during late gestation.

According to the present invention, adjunctive treatment of swine withThyex (or Thyex plus antimicrobial agent) compositions enhances theefficacy of vaccine regimens in protecting PRRSV-susceptible respiratorysystems against PRRSV.

According to the present invention, adjunctive treatment of swine withThyex (or Thyex plus antimicrobial agent) compositions reducesvaccine-induced reproductive failure when such vaccines are administeredduring gestation, including late gestation.

Dose Determinations

A therapeutically-effective dose of a composition of the presentinvention refers to that amount of the composition sufficient to preventor inhibit the effects of the treated condition, or to that amountsufficient to enhance the efficacy of adjunctive regimens. This amountmay vary somewhat among subjects, but are nonetheless reasonablydetermined by one of ordinary skill within the art in view of the manyart-recognized symptoms associated with the treated conditions.

Therapeutically-effective doses of the disclosed compositions areadministered alone or in combination with other therapeutic agents, suchas macrophage stimulating agents, anti-microbial agents (e.g.,antiviral, antifungal or antibacterial agents), or are administered asadjunctive therapy in combination with administration of other treatmentregimens.

In particular aspects, as in the Examples herein, the Thyex compositionsare standardized at a protein concentration about 2 mg/ml. Preferably,the daily dose range for Thyex administration by injection is from about0.05 mg/kg to about 1 mg/kg. More preferably, the dose range for Thyexadministration by injection is from about 0.05 mg/kg to about 0.5 mg/kg.Even more preferably, the dose range for Thyex administration byinjection is from about 0.1 mg/kg to about 0.4 mg/kg. Most preferably,the dose range for Thyex administration by injection is from about 0.2mg/kg to about 0.3 mg/kg.

In particular aspects, the daily dose range for Thyex oraladministration is from about 1 mg/kg to about 20 mg/kg. More preferably,the dose range for Thyex oral administration is from about 1 mg/kg toabout 10 mg/kg. Even more preferably, the dose range for Thyex oraladministration is from about 3 mg/kg to about 9 mg/kg. Most preferably,the dose range for Thyex oral administration is from about 5 mg/kg toabout 8 mg/kg.

In particular aspects, the daily dose range for adjunctiveadministration of beta glucan can be determined by routine optimizationby one of ordinary skill in the art. In particular aspects, the dailydose range for adjunctive administration of the polysaccharide extract(e.g., consisting of about 70% beta 1-3 glucan and 30% tissue proteins)will be about 300 to about 500 mg per day for a typical patient (e.g.,or about 0.5 mg/kg to 15 mg/kg). In particular embodiments, using morehighly purified polysaccharide fractions (e.g., void of protein; e.g.,extracted by the method of Ohno et al (Biol Pharm Bul, 23, p 866, 2000),the daily dose will be about 300 mg per day (e.g., or about 0.5 mg/kg to2.0 mg/kg) for a typical patient.

Preferably, as in the Examples herein, the inventive heat-distilledHouttuynia cordata extract (DYXC-1 and 2) compositions are standardizedat a value of about 200 mg (dried herb wt equivalents; dhe)/ml.

Preferably, in terms of dried herb wt equivalents, the daily dose rangefor the inventive heat-distilled Houttuynia cordata extract (DYXC-1 and2) administration injection is from about 5 mg/kg to about 50 mg/kg.More preferably, the dose range for the heat-distilled Houttuyniacordata extract (DYXC-1 and 2) administration by injection is from about10 mg/kg to about 40 mg/kg. Even more preferably, the dose range for theheat-distilled Houttuynia cordata extract (DYXC-1 and 2) administrationby injection is from about 15 mg (dhe)/kg to about 30 mg (dhe)/kg. Mostpreferably, the dose range for the heat-distilled Houttuynia cordataextract (DYXC-1 and 2) administration by injection is from about 20 mg(dhe)/kg to about 25 mg (dhe)/kg.

Antibiotic dosages were those of the label, according to the particularantibiotic used.

Formulations and Use

In particular preferred aspects, Thyex-1-6A and -6B have substantialutility in methods for treatment of various Human and mammalianconditions including, but not limited to impaired physical vigor oraptitude, and aging and/or age-related conditions (arthritis, mobilitydeficits, loss of appetite, etc.), comprising administration of saidcompositions.

For administration by injection, the Thyex, and the heat-distilledHouttuynia cordata extract (DYXC-1 and 2) compositions of the presentinvention are preferably formulated in aqueous solutions withphysiologically compatible buffered saline (e.g., phosphate bufferedstandard physiological saline; 0.85% NaCl).

For oral administration, the pharmaceutical Thyex and the inventiveheat-distilled Houttuynia cordata extract (DYXC-1 and 2) compositions ofthe present invention may take the form of, for example, liquids, gels,syrups, slurries, and the like, prepared by conventional means withpharmaceutically acceptable excipients such as: binding agents (e.g.,pre-gelatinized maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP)); fillers (e.g., lactose, sucrose, mannitol,or sorbitol, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch, sodium starch glycolate, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate); or wetting agents (e.g., sodium lauryl sulfate). Such liquidpreparations are prepared by conventional means with pharmaceuticallyacceptable additives such as suspending agents (e.g., sorbitol syrup,cellulose derivatives or hydrogenated edible fats); emulsifying agents(e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, ethyl alcohol or fractionated vegetable oils); and preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Thepreparations may also contain buffer salts, flavoring, coloring andsweetening agents as appropriate.

Additional oral administration of the inventive heat-distilledHouttuynia cordata extract (DYXC-1 and 2) compositions can be in theform of an effervescent tablet. Effervescent formulations are known inthe art for various active ingredients and vitamins. These effervescentformulations generally include an agent which is capable of releasingCO₂, and an agent which induces the release of CO₂. Suitable agentscapable of releasing CO₂ which are used include alkali metal carbonatesor alkali metal bicarbonates, such as sodium carbonate and sodiumbicarbonate. Alkaline earth metal carbonate formulations are mainlycontained in mineral preparations. Suitable agents for inducing CO₂release include edible organic acids, or their acidic salts, which arepresent in solid form and which can be formulated with the activeingredient and the other auxiliaries to provide granules or tablets,without premature evolution of CO₂. The active ingredients are eitherpresent in the effervescent formulation as readily soluble compounds, orthey are solubilized by salt formation during the dissolution process.

For administration by inhalation, the Thyex and the heat-distilledHouttuynia cordata extract (DYXC-1 and 2) compositions for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

The Thyex, and the heat-distilled Houttuynia cordata extract (DYXC-1 and2) compositions of the present invention may be formulated forparenteral administration by injection by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform in ampoules or in multi-dose containers, with optionally, with anadded preservative.

Vaccines are either those commercially available, or those preparedaccording to art-recognized methods, and are of various forms, includingconventional forms such as aqueous dispersions, oil emulsions, liposomecompositions, lyophilized forms, etc. Vaccine compositions andvaccination regimens may comprise different adjuvants, emulsifiers,stabilizers, etc. Vaccines are administered by different routes,including but not limited to parenteral, intramuscular, intranasal,intratracheal, subcutaneous, cutaneous, percutaneous or intracutaneousroutes, and combinations thereof.

Vaccines may be prepared, inter alia, as aqueous solutions, syrups,elixers, or tinctures, and the liquid formulations may includesuspensions and/or emulsions.

Thyex-4 may be lyophilized and dispensed in “00” size gelatin capsules:Oral. Approximately 40% thymic polypeptides. According to particularaspects, Thyex-4 may be used for stimulation of immune functions (e.g.,anti-aging), to cure allergy and certain autoimmune disorders (e.g.,celiac disease); and for treating gout.

Thyex-5. In particular aspects, Thyex-5 (e.g., lyophilized;approximately 80% thymic polypeptides) is mixed with other extracts(e.g., extracts containing polysaccharides such as beta 1-3 glucan). Themixtures, for example, can be dispensed in “00” gelatin capsules, oralternatively, for example, in size “3” capsule if not mixed with otherextracts.

Thyex 6A. In particular aspects, Thyex-6A (e.g., sterile liquid extract)can be used to generate aerosols (e.g. for treating pneumonia oremphysema). Alternatively, for example, ointments can be used whenThyex-6A is mixed with water-soluble ointment base for treating, forexample, lichen sclerosis et atrophicus, and wounds.

Thyex 6B. In particular aspects, Thyex-6BA (e.g., sterile liquidbuffered, and saline adjusted for injection; at least 99% pure) is usedfor veterinary and human uses, including, but not limited to veterinaryuses including: arthritis, anti-aging; pneumonia; dust pneumonia (IV),single tumor mass, such as Cancer Eye (squamous cell carcinoma inwhite-faced Herefords); virus infections, such as distemper, PRRS,shipping fever, etc.; shock trauma; arthritis, etc., and Human usesincluding, but not limited to: autoimmune disorders (IV or SQ) such astype 1 diabetes, lupus, psoriasis, rheumatoid arthritis, etc.; seniledementia, etc.

In addition to the ingredients specifically mentioned above, theformulations of the present invention may include other agents known tothose skilled in the art, having regard for the type of formulation inissue. For example, formulations suitable for oral administration mayinclude flavoring agents and formulations suitable for intranasaladministration may include perfumes.

The therapeutic compositions of the invention can be administered by anyconventional method available for use in conjunction with pharmaceuticaldrugs, either as individual therapeutic agents or in a combination oftherapeutic agents.

The dosage administered will, of course, vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent and its mode and route of administration; the age, health andweight of the recipient; the nature and extent of the symptoms; the kindof concurrent treatment; the frequency of treatment; and the effectdesired.

The injection-use formulations can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials, and can bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid excipient, or the heat-distilledHouttuynia cordata extract (DYXC-1 and 2), immediately prior to use.Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules, and tablets. The requirements for effectivepharmaceutical carriers for injectable compositions are well known tothose of ordinary skill in the art. See, for example, Pharmaceutics andPharmacy Practice, J. B. Lippincott Co., Philadelphia, Pa., Banker andChalmers, Eds., 238-250 (1982) and ASHP Handbook on Injectable Drugs,Toissel, 4th ed., 622-630 (1986).

Formulations suitable for topical administration include lozengescomprising a heat-distilled Houttuynia cordata extract (DYXC-1 and 2) ofthe invention and optionally, an additional therapeutic and a flavor,usually sucrose and acacia or tragacanth; pastilles comprising agas-enriched fluid and optional additional therapeutic agent in an inertbase, such as gelatin and glycerin, or sucrose and acacia; and mouthwashes or oral rinses comprising a gas-enriched fluid and optionaladditional therapeutic agent in a suitable liquid carrier; as well ascreams, emulsions, gels, and the like.

Additionally, formulations suitable for rectal administration may bepresented as suppositories by mixing with a variety of bases such asemulsifying bases or water-soluble bases. Suitable pharmaceuticalcarriers are described in Remington's Pharmaceutical Sciences, MackPublishing Company, a standard reference text in this field.

The dose administered to a subject, especially an animal, particularly ahuman, in the context of the present invention should be sufficient toeffect a therapeutic response in the animal over a reasonable timeframe. One skilled in the art will recognize that dosage will dependupon a variety of factors including the condition of the animal, thebody weight of the animal, as well as the condition being treated. Asuitable dose is that which will result in a concentration of thetherapeutic composition in a subject that is known to affect the desiredresponse.

The size of the dose also will be determined by the route, timing andfrequency of administration as well as the existence, nature, and extentof any adverse side effects that might accompany the administration ofthe therapeutic composition and the desired physiological effect.

Most suitable means of administration for a particular subject willdepend on the nature and severity of the disease or condition beingtreated or the nature of the therapy being used, as well as the natureof the therapeutic composition or additional therapeutic agent. Incertain embodiments, oral or topical administration is preferred.

Formulations suitable for oral administration may be provided asdiscrete units, such as tablets, capsules, cachets, syrups, elixirs,chewing gum, “lollipop” formulations, microemulsions, solutions,suspensions, lozenges, or gel-coated ampules, each containing apredetermined amount of the active compound; as powders or granules; assolutions or suspensions in aqueous or non-aqueous liquids; or asoil-in-water or water-in-oil emulsions.

In addition to the ingredients specifically mentioned above, theformulations of the present invention may include other agents known tothose skilled in the art, having regard for the type of formulation inissue. For example, formulations suitable for oral administration mayinclude flavoring agents and formulations suitable for intranasaladministration may include perfumes.

The therapeutic compositions of the invention can be administered by anyconventional method available for use in conjunction with pharmaceuticaldrugs, either as individual therapeutic agents or in a combination oftherapeutic agents.

Example 1 Preparation of Thymus Extracts Thyex-1

This example provides an exemplary process embodiment used for preparingthymus extracts, and compositions (“Thyex-1”) produced in accordancetherewith:

Thyex-1:

Thyex-1 Process.

The following steps (1)-(16) comprise a process embodiment for producingThyex-1 (step (17) relates to storage) suitable for oral delivery:

(1) Homogenization of Thymus Tissue.

Fresh “prime” (i.e., not fibrous or whitish in appearance) porcine orbovine thymus glands were frozen (e.g., overnight). The frozen glandswere rinsed briefly in clean water and “dressed” by removal of anyassociated fibrous or connective tissue, fatty tissue, or lymph nodetissue. The prime washed, dressed thymus glands were cut into smallpieces (e.g., about 2″ cubes using a sharp knife), and homogenizedthrough the use of a food processor or other grinding apparatus (e.g., ameat grinder). For homogenization, a volume of approximately 700 ml of0.2% NaCl solution (in distilled water) was blended with approximately350 g wet weight (about 400 ml wet volume) of cut-up thymus tissue in astandard size blender for at least one minute to produce a thymushomogenate;

(2) Low-Speed Centrifugation.

The “thymus homogenate” of step (1) was centrifuged at about 3,500×G for10 minutes at ambient temperature to produce a pellet and a supernatantfraction;

(3) Crude Filtration.

The resulting “supernatant fraction” of step (2) (after removal of anypacked low density debris floating on its surface) was decanted from thecentrifugation pellet and gravity filtered through one or more layers ofstandard cheese cloth to produce a primary filtered supernatant;

(4) Production of a “Secondary Filtered Supernatant.”

Steps (1)-(3) were repeated with another 350 g wet weight (about 400 mlwet volume) of prime washed, dressed, cut-up thymus glands, except thatthe “primary filtered supernatant” of step (3) was used in place of the700 ml of 0.2% NaCl solution of step (1). This substitution allowed forthe production of a more concentrated (relative to the “primary filteredsupernatant”) secondary filtered supernatant;

(5) Heat Denaturation.

The “secondary filtered supernatant” of step (4) was heated to atemperature of about 75-80° C. by exposing the container thereof to auniform heat source, such as a constant temperature water bath set atabout 100° C., or a double boiler containing water at about 100° C.During said heating, the “secondary filtered supernatant” was frequentlyagitated or stirred until it reached about 75-80° C. to produce aheat-denatured secondary filtered supernatant;

(6) Low-Speed Centrifugation.

The “heat-denatured secondary filtered supernatant” of step (5) wascentrifuged at 3,500×g for 5 minutes at ambient temperature to produce apellet and a heat-denatured supernatant fraction;

(7) Production of a “Heat-Denatured Filtered Supernatant.”

The “heat-denatured supernatant fraction” of step (6) was decanted fromthe centrifugation pellets and gravity filtered through one or morelayers of standard cheese cloth to produce a filtered, heat-denaturedsupernatant fraction (hereinafter the “intermediate supernatant”fraction) that was still slightly warm from the heat denaturation ofstep (5);

(8) Ammonium Sulfate Precipitation.

About 650 gm of ammonium sulfate was added to 1 L of the warm“intermediate supernatant” of step (7). The solution was stirred untilall the ammonium sulfate was dissolved, and then allowed to stand forabout 1 hour at ambient temperature to produce a salted intermediatesupernatant fraction;

(9) Low-Speed Centrifugation.

The “salted intermediate supernatant” of step (8) was divided betweentwo, 1 L centrifuge bottles and centrifuged at 3,500×g for 10 minutes atambient temperature to produce ammonium sulfate pellets, and supernatantfractions;

(10) Suspension of Ammonium Sulfate Pellet Fraction.

The “ammonium sulfate supernatants” from step (9) were decanted from thecentrifugation tubes and discarded, and excess salt solution wascarefully wiped from the inside tube walls. The two ammonium sulfatepellets of step (9) (i.e., corresponding to each 1-L centrifuge bottle)were then suspended and dissolved by gentle mixing with about 50 ml of0.01 to 0.05 M phosphate buffer (about pH 7) for each pellet(alternatively, the pellets were suspended with distilled water). Thesuspensions were allowed to stand for about 1 hour at ambienttemperature with brief agitation about every 15 minutes (to facilitatecomplete dissolution of the pellets) to provide an ammonium sulfatefraction. Note that dissolution of any remaining ammonium sulfate pelletcan be affected by the step-wise addition of small amounts of distilledwater (e.g., 5 ml aliquots), followed by agitation until the pellet iscompletely dissolved;

(11) Dialysis.

The “ammonium sulfate” fraction of step (10) was transferred to cleandialysis tubing (e.g., Spectrapor 3.5 kDa molecular weight cut-offsize), and dialyzed with stirring (e.g., by means of amagnetically-driven stir bar in the dialysis chamber) for 3 days againstan excess of distilled water at about 4° C. to produce a dialyzedammonium sulfate fraction. The distilled water was changed every 12hours. Increasing hydrostatic pressure within the dialysis tubing wasperiodically relieved by removing some of the dialysate and transferringit to additional dialysis tubes;

(12) High-Speed Centrifugation.

The “dialyzed ammonium sulfate fraction” of step (11) was centrifuged at8,500×g for 10 minutes at ambient temperature to produce a pellet anddialyzed ammonium sulfate supernatant fraction;

(13) First Exclusion-Membrane Filtration.

The “dialyzed ammonium sulfate supernatant fraction” of step (12) waspassed under nitrogen pressure at about 40-50 p.s.i. through a 100 kDaexclusion limit membrane filter (Amicon) at 4° C. (alternatively,ambient temperature will suffice) to produce a 3.5 kDa to 100 kDafiltrate;

(14) Second Exclusion-Membrane Filtration.

The “3.5 kDa to 100 kDa filtrate” of step (13) was passed under nitrogenpressure at 40 to 50 p.s.i. (275.8 to 344.75 Kpa, in metric units)through a 30 kDa exclusion limit membrane filter (Amicon) to produce a3.5 kDa to 30 kDa filtrate;

(15) Adjustment of pH and Ionic Strength.

About 5 ml of 1 M phosphate buffer (about pH 7) per liter was added tothe “3.5 kDa to 30 kDa filtrate” of step (14). Solid NaCl was then addedto 0.85% (weight to volume) to produce a pH- and ionic strength-adjusted30 kDa filtrate, Thyex-1;

(16) Filter Sterilization.

The “Thyex-1” of step (15) was filter sterilized by passage through a0.2μ membrane filter to produce sterile Thyex-1, suitable for oraldelivery or delivery; and

(17) Storage.

Thyex-1, produced in accordance with steps (1)-(16) of the Thyex-1process, was typically stored frozen (e.g., −5° C. to −20° C.) insterilized containers, and thawed just prior to use. According toparticular aspects, the therapeutic activity of Thyex-1 was found to bestable to repeated freezing and thawing. Alternatively, Thyex-1 waslyophilized, stored at ambient temperature and reconstituted withsterile water prior to use.

According to particular aspects, one or more of the above steps areoptional.

Example 2 Preparation of Thymus Extracts Thyex-2

This example provides an exemplary process embodiment used for preparingthymus extracts, and compositions (“Thyex-2”) produced in accordancetherewith suitable for oral delivery:

Thyex-2:

Thyex-2 Process.

The following steps (1)-(8) comprise a process embodiment for producingThyex-2 (step (9) relates to storage):

(1) Production of “Intermediate Supernatant.”

Steps (1)-(7) of the above-identified Thyex-1 process for thepreparation of “intermediate supernatant” were followed (except thatsteps (1)-(3) were not repeated as in the Thyex-1 process) to produce an“intermediate supernatant” fraction;

(2) High-Speed Centrifugation.

The “intermediate supernatant” fraction of step (1) was cleared (i.e.,to remove potential pathogens) by centrifugation at 8,500×g for 10minutes at ambient temperature to produce a pellet and a clearedintermediate supernatant fraction;

(3) Lyophilization.

The “cleared intermediate supernatant” fraction of step (2) waslyophilized (i.e., freeze dried) either to complete dryness to produce adried, cleared intermediate supernatant fraction, or until its volumewas reduced by 90% to produce a lyophilized, cleared intermediatesupernatant fraction;

(4) Dialysis.

The “lyophilized, cleared intermediate supernatant,” or the alternativecompletely “dried” fraction (suspended in 500 ml distilled water per13.6 kg (30 lbs.) wet weight of thymus glands processed) of step (3) wasdialyzed according to step (11) of the above-identified Thyex-1 processto produce a dialyzed, lyophilized intermediate supernatant fraction;

(5) High-Speed Centrifugation.

The “dialyzed, lyophilized intermediate supernatant” of step (4) wascentrifuged at 8,500×g for 10 minutes at ambient temperature to producea pellet, and a cleared, dialyzed, lyophilized intermediate supernatantfraction;

(6) Exclusion-Membrane Filtration.

The “cleared dialyzed, lyophilized intermediate supernatant” of step (5)was passed consecutively under nitrogen pressure (40-50 p.s.i.) through100 kDa and 30 kDa exclusion limit membrane filters (Amicon), accordingto steps (13) and (14) of the above-identified Thyex-1 process toproduce a 3.5 kDa to 30 kDa filtrate. The protein concentration of the“30 kDa filtrate” was measured, and optionally diluted (typically, toabout 2 mg/0.25 ml (lesser or greater dilutions were also made asdesired);

(7) Adjustment of pH and Ionic Strength.

The pH and ionic strength of the “3.5 kDa to 30 kDa filtrate” or theoptionally diluted “3.5 kDa to 30 kDa filtrate” of step (6) was adjustedaccording to step (15) of the above-identified Thyex-1 process toproduce a pH- and ionic strength-adjusted 3.5 kDa to 30 kDa filtrate,Thyex-2;

(8) Filter Sterilization.

The “Thyex-2” of step (7) was filter sterilized according to step (16)of the above-identified Thyex-1 process to produce sterile Thyex-2,suitable for oral delivery; and

(9) Storage.

Thyex-2, produced in accordance with steps (1)-(8) of the Thyex-2process was typically stored frozen (e.g., −5° C. to −20° C.) insterilized containers, and thawed just prior to use. According toparticular aspects, the therapeutic activity of Thyex-2 was found to bestable to repeated freezing and thawing. Alternatively, Thyex-2 waslyophilized, stored at ambient temperature and reconstituted withsterile water prior to use.

According to particular aspects, one or more of the above steps areoptional.

Example 3 Preparation of Thymus Extracts Thyex-3

This example provides an exemplary process embodiment used for preparingthymus extracts, and compositions (“Thyex-3”) produced in accordancetherewith suitable for oral delivery:

Thyex-3:

Thyex-3 Process.

The following steps (1)-(10) comprise a process embodiment for producingThyex-3 (step (11) relates to storage), suitable for oral delivery:

(1) Homogenization of Thymus Tissue.

Fresh “prime” (i.e., not fibrous or whitish in appearance) porcine orbovine thymus glands were frozen (e.g., overnight). The frozen glandswere rinsed briefly in clean water and “dressed” by removal of anyassociated fibrous or connective tissue, fatty tissue, or lymph nodetissue. The prime washed, dressed thymus glands were cut into smallpieces (e.g., about 2″ cubes using a sharp knife), and homogenizedthrough the use of a food processor or other grinding apparatus (e.g., ameat grinder). For homogenization, a volume of approximately 800 ml of0.2% NaCl solution (in distilled water) was blended with approximately300 g wet weight (about 340 ml wet tissue volume) of cut-up thymustissue in a standard size blender for at least one minute to produce athymus homogenate;

(2) Low-Speed Centrifugation.

The “thymus homogenate” of step (1) was centrifuged at about 3,500 rpmfor 10 minutes at ambient temperature to produce a pellet and asupernatant fraction;

(3) Crude Filtration.

The resulting “supernatant fraction” of step (2) (after removal of anypacked low density debris floating on its surface) was decanted from thecentrifugation pellet and gravity filtered through one or more layers ofstandard cheese cloth to produce a primary filtered supernatant;

(4) Production of a “Secondary Filtered Supernatant.”

Steps (1)-(3) were repeated with another 175 g wet weight (200 ml wettissue volume) of prime washed, dressed, cut-up thymus glands, exceptthat the “primary filtered supernatant” of step (3) was used in place ofthe 800 ml of 0.2% NaCl solution of step (1). This substitution allowedfor the production of a more concentrated (relative to the “primaryfiltered supernatant”) secondary filtered supernatant;

(5) Production of a “Tertiary Filtered Supernatant.”

Steps (1)-(3) were repeated with another 200 ml (wet volume) of primewashed, dressed, cut-up thymus glands, except that the “secondaryfiltered supernatant” from step (4) was used in place of the 800 ml of0.2% NaCl solution of step (1). This substitution allowed for theproduction of a more concentrated (relative to the “primary” and“secondary filtered supernatants”) tertiary filtered supernatant;

(6) Heat Denaturation.

The “tertiary filtered supernatant” from step (5) was heated to atemperature of about 75-80° C. by exposing the container thereof to auniform heat source such as a constant-temperature water bath set atabout 100° C. or a double boiler containing water at about 100° C.During heating, the “tertiary filtered supernatant” was frequentlyagitated or stirred until it reached about 75-80° C. to produce aheat-denatured tertiary filtered supernatant fraction;

(7) Low-Speed Centrifugation.

The “heat-denatured tertiary filtered supernatant” fraction of step (6)was centrifuged at 3,500 rpm for 5 minutes at ambient temperature toproduce a pellet and a heat-denatured supernatant fraction;

(8) Production of a “Heat-Denatured Filtered Supernatant.”

The “heat-denatured supernatant fraction” of step (7) was decanted fromthe centrifugation pellets and gravity filtered through one or morelayers of standard cheese cloth to produce a filtered, heat-denaturedsupernatant fraction that was still slightly warm from the heatdenaturation of step (6);

(9) High-Speed Centrifugation.

The “filtered, heat-denatured supernatant” fraction of step (8) wascentrifuged at about 8,500×g for 5 minutes at ambient temperature toproduce a pellet, and a high-speed supernatant fraction, Thyex-3;

(10) Filter Sterilization.

The “Thyex-3” fraction of step (9) was filter sterilized according tostep (16) of the above-identified Thyex-1 process to produce sterileThyex-3, suitable for oral delivery; and

(11) Storage.

Thyex-3, produced in accordance with steps (1)-(10) of the Thyex-3process was typically stored frozen (e.g., −5 to −20° C.) in sterilizedcontainers, and thawed just prior to use. According to particularaspects, the therapeutic activity of Thyex-3 was found to be stable torepeated freezing and thawing. Alternatively, Thyex-3 was lyophilized,stored at ambient temperature and reconstituted with sterile water priorto use.

According to particular aspects, one or more of the above steps areoptional.

Example 4 Preparation of Thymus Extracts Thyex-4

With reference to FIG. 4, this example provides an exemplary processembodiment used for preparing thymus extracts, and compositions(“Thyex-4”) produced in accordance therewith suitable for oral delivery:

Thyex-4:

Thyex-4 Process.

The following steps (1)-(11) comprise a process embodiment for producingThyex-4 (step (12) relates to storage), e.g., suitable for oral delivery(NOTE: the following steps (1)-(6) are referred to herein as “stage 1steps (1)-(6)”:

Stage 1 Steps (1)-(6):

(1) Homogenization of Thymus Tissue.

Fresh “prime” (e.g., preferably not fibrous or whitish in appearance)porcine ovine or bovine thymus glands were frozen (e.g., overnight, orin some instances, preferably for at least 72 hours). The frozen glandswere rinsed briefly in clean water and “dressed” by removal of anyassociated fibrous or connective tissue, fatty tissue, or lymph nodetissue. The prime washed, dressed thymus glands were cut or minced intosmall pieces (e.g., about 1″ to about 2″ cubes using a sharp knife), andhomogenized through the use of a food processor or other grindingapparatus (e.g., a meat grinder). For homogenization, a volume ofapproximately 700 ml of 0.2% NaCl solution (in distilled water) wasblended for at least a minute with approximately 350 g wet weight ofcut-up thymus tissue in a standard size blender to produce a thymushomogenate;

(2) Low-Speed Centrifugation.

The “thymus homogenate” of step (1) was centrifuged at about 3,500 rpmfor 10 minutes at ambient temperature to produce a pellet and asupernatant fraction;

(3) Crude Filtration.

The resulting “supernatant fraction” of step (2) (after removal of anypacked low density debris floating on its surface) was decanted from thecentrifugation pellet and gravity filtered through one or more layers ofstandard cheese cloth to produce a primary filtered supernatant;

(4) Heat Denaturation.

The “primary filtered supernatant” of step (3) was heated to atemperature of about 75-80° C. by exposing the container thereof to auniform heat source, such as a constant temperature water bath set atabout 100° C., or a double boiler containing water at about 100° C.During said heating, the “primary filtered supernatant” was frequentlyagitated or stirred until it reached about 75-80° C. to produce aheat-denatured primary filtered supernatant;

(5) Low-Speed Centrifugation.

The “heat-denatured primary filtered supernatant” of step (4) wascentrifuged at 3,500×g for 5 minutes at ambient temperature to produce apellet and a heat-denatured supernatant fraction;

(6) Production of a “Heat-Denatured Filtered Supernatant.”

The “heat-denatured supernatant fraction” of step (5) was decanted fromthe centrifugation pellets and gravity filtered through one or morelayers of standard cheese cloth to produce a filtered, heat-denaturedsupernatant fraction (hereinafter the “intermediate supernatant”fraction) that was still slightly warm from the heat denaturation ofstep (4);

Stage 2 Steps (7)-(6):

(7) Dialysis.

The “intermediate supernatant” fraction of step (6) was dialyzedaccording to step (11) of the above-identified Thyex-1 process (e.g.,using clean dialysis tubing (e.g., Spectrapor 3.5 kDa molecular weightcut-off size), and dialyzed with stirring (e.g., by means of amagnetically-driven stir bar in the dialysis chamber) for 3 days againstan excess of distilled water at about 4° C.) to produce a dialyzed,intermediate supernatant fraction;

(8) Low-Speed Centrifugation.

The “dialyzed, intermediate supernatant fraction” of step (7) wascentrifuged at 3,500 rpm for 5 minutes at ambient temperature to producea pellet and a heat-denatured supernatant fraction;

(9) Production of a “Heat-Denatured Filtered Supernatant.”

The “heat-denatured supernatant fraction” of step (8) was decanted fromthe centrifugation pellets and gravity filtered through one or morelayers of standard cheese cloth to produce a filtered, heat-denaturedsupernatant fraction;

(10) High-Speed Centrifugation.

The “filtered, heat-denatured supernatant” fraction of step (9) wascentrifuged at about 8,500×g for 5 minutes at ambient temperature toproduce a pellet, and a high-speed supernatant fraction, Thyex-4;

(11) Filter Sterilization.

The “Thyex-4” fraction of step (10) was filter sterilized according tostep (16) of the above-identified Thyex-1 process to produce sterileThyex-4, suitable for oral delivery; and

(12) Storage.

Thyex-4, produced in accordance with steps (1)-(11) of the Thyex-4process was typically stored frozen (e.g., −5 to −20° C.) in sterilizedcontainers, and thawed just prior to use. According to particularaspects, the therapeutic activity of Thyex-4 is stable to repeatedfreezing and thawing. Alternatively, Thyex-4 was lyophilized, stored atambient temperature and reconstituted with sterile water prior to use.

According to particular aspects, one or more of the above steps areoptional.

Example 5 Preparation of Thymus Extracts Thyex-5

With reference to FIG. 4, this example provides an exemplary processembodiment used for preparing thymus extracts, and compositions(“Thyex-5”) produced in accordance therewith suitable for oral delivery:

Thyex-5:

Thyex-5 Process.

The following steps (1)-(13) comprise a process embodiment for producingThyex-5 (step (14) relates to storage), suitable for oral delivery:

(1)-(6) (see Stage 1 steps (1)-(6) for Thyex-4 above);

(7) Ammonium Sulfate Precipitation.

About 750 to about 800 gm of ammonium sulfate was added to 1 L of thewarm “intermediate supernatant” of step (6). The solution was stirreduntil all the ammonium sulfate was dissolved, and then allowed to standfor about 1 hour at ambient temperature to produce a salted intermediatesupernatant fraction;

(8) Low-Speed Centrifugation.

The “salted intermediate supernatant” of step (7) was divided betweentwo, 1 L centrifuge bottles and centrifuged at 3,500×g for 10 minutes atambient temperature to produce ammonium sulfate pellets, and supernatantfractions;

(9) Suspension of Ammonium Sulfate Pellet Fraction.

The “ammonium sulfate supernatants” from step (8) were decanted from thecentrifugation tubes and discarded, and excess salt solution wascarefully wiped from the inside tube walls. The two ammonium sulfatepellets of step (8) (i.e., corresponding to each 1-L centrifuge bottle)were then suspended and dissolved by gentle mixing with about 50 ml ofdistilled water (or optionally with 0.01 to 0.05 M phosphate buffer(about pH 7)) for each pellet. The suspensions were allowed to stand forabout 1 hour at ambient temperature with brief agitation about every 15minutes (to facilitate complete dissolution of the pellets) to providean ammonium sulfate fraction. Note that dissolution, if desired, of anyremaining ammonium sulfate pellet can be affected by the step-wiseaddition of small amounts of distilled water (e.g., 5 ml aliquots),followed by agitation until the pellet is completely dissolved;

(10) Dialysis.

The “ammonium sulfate” fraction of step (9) was transferred to cleandialysis tubing (e.g., Spectrapor 3.5 kDa molecular weight cut-offsize), and dialyzed with stirring (e.g., by means of amagnetically-driven stir bar in the dialysis chamber) for 3 days againstan excess of distilled water at about 4° C. to produce a dialyzedammonium sulfate fraction. The distilled water was changed every 12hours. Increasing hydrostatic pressure within the dialysis tubing wasperiodically relieved by removing some of the dialysate and transferringit to additional dialysis tubes;

(11) High-Speed Centrifugation.

The “dialyzed ammonium sulfate fraction” of step (10) was centrifuged at8,500×g for 10 minutes at ambient temperature to produce a pellet anddialyzed ammonium sulfate supernatant fraction (Thyex-5);

(12) Adjustment of pH and Ionic Strength.

Optionally, about 5 ml of 1 M phosphate buffer (about pH 7) per liter isadded to the “dialyzed ammonium sulfate supernatant fraction of step(11). Optionally, solid NaCl is then added to 0.85% (weight to volume)to produce a pH- and ionic strength-adjusted dialyzed ammonium sulfatesupernatant fraction (Thyex-5);

(13) Filter Sterilization.

The “Thyex-5” of step (12) was filter sterilized by passage through a0.2μ membrane filter to produce sterile Thyex-5, suitable for oraldelivery; and

(14) Storage.

Thyex-5, produced in accordance with steps (1)-(13) of the Thyex-5process, was typically stored frozen (e.g., −5° C. to −20° C.) insterilized containers, and thawed just prior to use. According toparticular aspects, the therapeutic activity of Thyex-5 was found to bestable to repeated freezing and thawing. Alternatively, Thyex-5 waslyophilized, stored at ambient temperature and reconstituted withsterile water prior to use.

According to particular aspects, one or more of the above steps areoptional.

Above steps (7)-(11) are referred to herein as Stage 2 steps (7)-(11).

Example 6 Preparation of Thymus Extracts Thyex-6A

Thyex-6A.

With reference to FIG. 4, this example provides an exemplary processembodiment used for preparing thymus extracts, and compositions(“Thyex-6A”) produced in accordance therewith suitable for oraldelivery, or delivery as a topical ointment or by injection orinhalation:

Thyex-6A:

Thyex-6A Process.

The following steps (1)-(14) comprise a process embodiment for producingThyex-6A (step (15) relates to storage), suitable for oral delivery:

(1)-(6) (see Stage 1 steps (1)-(6) for Thyex-4 and Thyex-5 above);

(7)-(11) (see Stage 2 steps (7)-(11) for Thyex-5 above);

(12) First Exclusion-Membrane Filtration.

The “dialyzed ammonium sulfate supernatant fraction” of step (11) waspassed under nitrogen pressure at about 40-50 p.s.i. through a 100 kDaexclusion limit membrane filter (Amicon) at 4° C. (alternatively,ambient temperature will suffice) to produce a 3.5 kDa to 100 kDafiltrate;

(13) Second Exclusion-Membrane Filtration.

The “3.5 kDa to 100 kDa filtrate” of step (12) was passed under nitrogenpressure at 40 to 50 p.s.i. (275.8 to 344.75 Kpa, in metric units)through a 30 kDa exclusion limit membrane filter (Amicon) to produce a3.5 kDa to 30 kDa filtrate (Thyex-6A);

(14) Filter Sterilization.

The “Thyex-6A” of step (13) was filter sterilized by passage through a0.2μ membrane filter to produce sterile Thyex-6A, suitable for oraldelivery, or delivery as a topical ointment or by injection orinhalation; and

(15) Storage.

Thyex-6A, produced in accordance with steps (1)-(14) of the Thyex-6Aprocess, was typically stored frozen (e.g., −5° C. to −20° C.) insterilized containers, and thawed just prior to use. According toparticular aspects, the therapeutic activity of Thyex-6A was found to bestable to repeated freezing and thawing. Alternatively, Thyex-6A waslyophilized, stored at ambient temperature and reconstituted withsterile water prior to use.

According to particular aspects, one or more of the above steps areoptional.

Example 7 Preparation of Thymus Extracts Thyex-6B

Thyex-6B Process.

With reference to FIG. 4, this example provides an exemplary processembodiment used for preparing thymus extracts, and compositions(“Thyex-6B”) produced in accordance therewith suitable for oraldelivery, or delivery as a topical ointment or by injection orinhalation:

Thyex-6B:

Thyex-6B Process.

The following steps (1)-(15) comprise a process embodiment for producingThyex-6A (step (16) relates to storage), suitable for oral delivery, ordelivery as a topical ointment or by injection or inhalation:

(1)-(6) (see Stage 1 steps (1)-(6) for Thyex-4, -5 and -6A above);

(7)-(11) (see Stage 2 steps (7)-(11) for Thyex-5 and -6A above);

(12) First Exclusion-Membrane Filtration.

The “dialyzed ammonium sulfate supernatant fraction” of step (11) waspassed under nitrogen pressure at about 40-50 p.s.i. through a 100 kDaexclusion limit membrane filter (Amicon) at 4° C. (alternatively,ambient temperature will suffice) to produce a 3.5 kDa to 100 kDafiltrate;

(13) Second Exclusion-Membrane Filtration.

The “3.5 kDa to 100 kDa filtrate” of step (12) was passed under nitrogenpressure at 40 to 50 p.s.i. (275.8 to 344.75 Kpa, in metric units)through a 30 kDa exclusion limit membrane filter (Amicon) to produce a3.5 kDa to 30 kDa filtrate (Thyex-6B);

(14) Adjustment of pH and Ionic Strength.

Optionally, about 5 ml of 1 M phosphate buffer (about pH 7) per liter isadded to the “dialyzed ammonium sulfate supernatant fraction of step(13). Optionally, solid NaCl is then added to 0.85% (weight to volume)to produce a pH- and ionic strength-adjusted dialyzed ammonium sulfatesupernatant fraction (Thyex-6B);

(15) Filter Sterilization.

The “Thyex-6B” of step (14) was filter sterilized by passage through a0.2μ membrane filter to produce sterile Thyex-6B, suitable for oraldelivery, or delivery as a topical ointment or by injection orinhalation; and

(16) Storage.

Thyex-6B, produced in accordance with steps (1)-(15) of the Thyex-6Bprocess, was typically stored frozen (e.g., −5° C. to −20° C.) insterilized containers, and thawed just prior to use. According toparticular aspects, the therapeutic activity of Thyex-6B was found to bestable to repeated freezing and thawing. Alternatively, Thyex-6B waslyophilized, stored at ambient temperature and reconstituted withsterile water prior to use.

According to particular aspects, one or more of the above steps areoptional.

Example 8 General Considerations for Practice of the Above-IdentifiedThyex 1-6A and -6B Process Embodiments

This Example 8 provides general considerations for practice of theabove-identified Thyex 1-6A and -6B process embodiments.

The above-described embodiments (Thyex-1 (steps 1-16), Thyex-2 (steps1-8), Thyex-3 (steps 1-10), Thyex-4 (steps 1-11), Thyex-5 (steps 1-13),Thyex-6A (steps 1-14) and Thyex 6B (steps 1-15)), and the storage (e.g.,lyophilization) steps of the inventive processes may be practiced withvarious modifications (including but not limited to those outlinedbelow) that are within the scope of the present invention, and withalternatives or substitutions that will be recognized by those ofordinary skill in the art as being equivalent to those used herein toproduce Thyex 1-6A and -6B.

Thymus Glands.

In particular aspects, animals (e.g., steers) are taken to a packinghouse at about 12-14 months. The thymus gland at this age is grayish. Asan animal ages, the gland begins to become fibrous and even whitish incolor. The optimum yield of final product from one kilogram (about 4.5lbs) of prime gland is 1 gram of purified Thyex (e.g., Thyex 6A or 6B).Sheep glands are generally from 6 month-old animals.

Freshly harvested thymus glands from porcine, ovine, or bovine sourcesshould optimally be frozen within 24 hours of harvest and stored frozen,preferably for at least 72 hours. Freezing of the thymus glands rendersthe cells more susceptible to disruption in isotonic salt solution(e.g., 0.2% to 0.3% salt, such as NaCl) during homogenization.Variations in the freezing temperature and duration are within the scopeof the present invention. The thymus glands are preferably frozen atleast once (e.g., −5 to −20° C.) for production of optimal extracts.

For example, to process, thawed glands are preferably first washed andextraneous materials, such as fatty tissues, lymph nodes, and connectivetissues are preferably excised and discarded. The tissues are preferablyminced into approximately 1″ squares before subjected to grinding (e.g.,in a food processor, meat grinder, blender, or equivalent or suitabledevice). Preferably, the ground glands are homogenized in a blender at aproportion of about 350 gm wet weight with 700 ml of 0.2% saline for atleast a minute. Following centrifugation (e.g., about 3,500×g for 10minutes), the supernatant solution (upper and lower ppt discarded) isheat denatured by raising the solution temperature in a double boilerwith constant stirring to a temperature in the range of about 75° C. to80° C. (preferably 75° C.). Following a second centrifugation at, e.g.,the same speed but for 5 minutes, the supernatant solution is collectedand precipitate (ppt) discarded. In particular embodiments, the glandsfor all Thyex processes 1, 2, 3, 4, 5, 6A, and 6B are processed throughthis phase in identical or very similar fashion.

Production of “secondary”- and “tertiary”-filtered supernatants, such asthose described in step (4) of the Thyex-1 process embodiment, step (1)of the Thyex-2 process embodiment, or steps (4) and (5) of the Thyex-3process embodiment, allows for more concentrated filtered supernatants(relative to the corresponding “primary”-filtered supernatants), thusreducing the amount of ammonium sulfate required (Thyex-1 processembodiment), or the lyophilization time required (Thyex-2 processembodiment) to process a given amount of thymus tissue. Generally,variations in the final protein concentrations (e.g., in the range of 1to 7 mg/ml) of the various primary-, secondary- and tertiary-filteredsupernatants reflect the average age of the animals from which thymustissue is obtained. Preferably, the protein concentration of thetertiary-filtered supernatant is about 4 mg/ml.

A heat-denaturation step is integral to all of the above-described Thyexprocess embodiments, and facilitates precipitation and subsequentremoval of relatively large, heat-labile proteins that have no utilityin the claimed compositions or methods (see below). Variation in thevolume of filtered supernatant fraction treated, in the finaltemperature of the heat-denaturation step (within the range of about 75°C. to about 80° C.), in the temperature of the uniform heat source(within the range of about 80° C. to about 100° C., preferably about100° C.) and in the time period over which heating of the filteredsupernatant fractions from initial to said final temperature takes place(generally within the range of about 5 to 20 minutes for a 1-litervolume of supernatant, but generally for lesser or greater periods oftime when heating smaller or larger volumes, respectively) are withinthe scope of the present invention. Preferably, the supernatant isheated to the final temperature at a rate that is as rapid as possiblewhereby said rate, in combination with stirring, generally minimizes theoccurrence of local supernatant temperatures (e.g., supernatanttemperatures near the heat-transferring wall of the supernatantcontainer) that exceed the desired final temperature.

Likewise, variations in the duration and frequency of stirring duringsaid heating are within the scope of the present invention, and dependon the temperature of the constant-temperature heat source and thevolume of supernatant being heated. Generally, both the duration andfrequency of stirring increase with increasing supernatant volume orheat-source temperature. Constant stirring is also effective, andpreferable when heating relatively large supernatant volumes.

Step (8) of the above-described Thyex-1 process embodiment, and step (7)of the above-described Thyex-5, -6A and -6B process embodiment involvesprotein concentration/fractionation by ammonium sulfate precipitation ofthe “intermediate supernatant” fraction. Most preferably, solid ammoniumsulfate is added to attain high salt concentrations (e.g., in excess ofabout 0.7 gm/ml) with minimal dilution. Alternatively, thisconcentration/fractionation step is achieved by adding saturatedammonium sulfate solution. However, because dilution of the intermediatesupernatant fraction is preferably minimized, this embodiment results inrelatively lower final salt concentrations (e.g., of about 0.5 gm/ml orgreater), and is thus less efficient in precipitating (and therebyrecovering) desirable low molecular weight proteins. Nonetheless,according to particular aspects, the resulting Thyex compositions haveactivity in the claimed methods, albeit to a lesser degree. Moreover,the present invention also encompasses the use of combinations ofsaturated or sub-saturated ammonium sulfate solutions with solidammonium sulfate.

A dialysis steps of the above-described Thyex process embodiments, allowany molecules of molecular weight less that about 3.5 kDa to passthrough. Variation in the precise exclusion limit of the dialysismembrane is within the scope of the present invention. Generally, anydialysis membrane is acceptable provided that its exclusion limit(porosity) enables the retention of molecules having molecular weightsof about 5 kDa or larger.

Additionally, variation in the precise exclusion limits of thefiltration membranes used in membrane filtrations steps of the Thyexprocess embodiments are within the scope of the present invention.Generally, any such filtration membrane is acceptable provided that itsexclusion limit (porosity) does not result in exclusion (i.e., removalfrom the final Thyex composition) of molecules having molecular weightsequal to or smaller than about 15 kDa. For example, exclusion membranesthat exclude molecules of about 20, 30 or 40 kDa or larger are useful inthe practice of the present invention, but result in final Thyexcompositions that are less active per mg of final protein, compared tothose compositions prepared using an exclusion membrane the excludesproteins larger than about 15 kDa. Preferably, dialysis and filtrationmembranes are chosen such that the resulting Thyex compositions compriseproteins in the molecular weight range of about 5 to 14 kDa.

The process embodiments (e.g., Thyex-3) may further comprisefractionation, based on molecular weight, to obtain a final proteinfraction having proteins of about 3.5 to about 30 kDa.

Many different types of membrane filters (e.g., cellulose acetatemembranes; Millipore) are commercially available for use in filtersterilization procedures. Some commercially-available membrane filtersare self-contained and provided as pre-sterilized, disposable units.Other membranes are mounted in reusable membrane holders, and heatsterilized in an autoclave prior to use.

Preferably, the final Thyex 1-6A and -6B compositions are standardizedat a protein concentration about 2 mg/ml, based on optical density at260 and 280 nm. Preferred dosages are discussed herein above under “DoseDeterminations.”

The instant processes comprise steps to optimize protein compositionsfor therapeutic use. For example, the above-described Thyex 6A and Thyex6B process embodiments are designed to provide therapeutic compositionssuitable for delivery as a topical ointment or by injection orinhalation, and include ammonium sulfate precipitation/fractionationsteps. Thyex-5 is prepared from a similar process but is somewhat lessrefined than Thyex-6A or Thyex-6B, and is designed to be preferablymixed in appropriate ratios with extracted lyophilized herbal sourcesand administered orally in, for example, filled gelatin capsules. TheThyex-4 process embodiment lacks ammonium sulfate precipitation step butprovides for a sufficiently-concentrated composition afterlyophilization. The resulting Thyex-4 composition is less refined inrelative to those of Thyex-5 (and Thyex-6A and -6B) but is nonethelesssuitably concentrated and formulated for efficacious oral delivery inboth animals and humans.

Example 9 Treatment of Aging and Related Conditions, and Restoration ofAthletic Vigor and Stamina Using the Inventive Thyex Compositions andCombinations Thereof

Overview.

All mammals possess a thymus gland at birth. As an animal ages, thegland begins to become fibrous and progressively degenerates. In humans,the thymus gland continues to grow until about age 20 beforedegenerating, and by age 50, no trace of glandular tissue is present.The progressive loss of the thymus can be temporally correlated to withdiminishing natural physical stamina, and increasing incidence ofage-related disorders.

According to additional aspects, the endocrine system is also involvedin the aging process, and the inventive Thyex compositions havesubstantial utility for additionally affecting aspects of the endocrinesystem, and have utility for treatment of aging and related conditions,and restoration of athletic vigor and stamina.

Viral Infections:

Particular observations relating to viral infections initially suggestedThyex modulation of the endocrine system. In one instance involving WeakCalf Syndrome, where afflicted newborn calves are too weak to rise/nurseand with mortality rate of over 95%, the treated animals were romping inabout 72 hours after a first Thyex infection followed by a secondinjection of Thyex 48 hours later. According to particular aspects, theresponse could not be explained simply due to stimulation of the immunesystem.

Similar observations and responses were seen by Applicant in pupsnaturally infected with canine distemper virus, which causes nearly 100%mortality rate. The surviving animals do not grow nor develop in sizeequal to that of uninfected animals in both situations. However, inlimited trials, regular scheduled injections with Thyex for both theafflicted calves and the afflicted pups resulted in the surviving younganimals to begin growing again.

Aging:

With respect to aging, several old dogs were treated with Thyex atregular schedules and showed signs of regaining youthfulness indicators,such as partial or complete relief from arthritic pain, increase inappetite and mobility, and in the case of one dog with defined atrophyof the hind quarters possibly due to lack of “exercise” associated witharthritis, the animal's hind quarters were significantly ‘filled’ withmuscle mass and the animal resumed daily jogging with its master.

According to additional aspects of the present invention, and withoutbeing bound by mechanism, these observations are explained, at least inpart, by implicating pituitary release of growth hormone. While thelevels of growth hormone in pituitary typically remains constantregardless of age, according to particular aspects, Thyex stimulates thehypothalamus to release growth-hormone-releasing hormone (whichapparently decreases as animal ages), which in turn stimulates pituitarygrowth hormone production.

According to particular aspects, regular treatment with Thyex acts torestore endocrine functions. In particular embodiments, an increase/risein libido was seen in women with ovarian tissues removed, and in elderlymen. In additional aspects, old uncastrated male dogs treated with Thyexshowed increased “mounting” tendencies.

The aging process has also linked to loss of a body's capacity tocontrol formation of free radicals and xeronine, which plays a majorrole in repairing many cell dysfunctions. Without being bound by theory,Thyex administration may restore such deficiencies.

Athletes:

It is a well-established fact that athletes injected with growth hormonehave “restored” physical vigor, as expected in a young person but lostas one ages. Thymic hormone has been reported to affect the endocrinesystem (e.g., release of FSH and LH in thymectomized mice by pituitaryresulting in production of testosterone/estrogen).

Applicants' observations indicate that Thyex directs the hypothalamus torelease hormones, in turn directing the pituitary to release specifichormones such as growth hormone, causing the liver to producesomatomedins, which are key hormones for regulating growth, increasedmetabolism, etc.

In particular aspects, individuals beyond 25 years in age are able todemonstrate physical aptitude, including recovery from physical stress,comparable individuals in their late teens.

Therefore, as an athlete ages there is a loss of vigor and/or stamina.According to particular aspects, and without being bound by theory,Thyex treatment benefits athletes, and particularly athletes in their30's and older, who can regain lost stamina, and improved recovery fromstressful exercises, etc.

According to particular aspects of the present invention, administeringthe inventive Thyex compositions to stimulate T cells, along with apolysaccharide extract to stimulate macrophage achieves a most effectiveimmune therapy response. In certain aspects, administration of Thyexalone is sufficient.

Beta Glucans.

Various investigators, including the present Applicant, have reportedthat administering BCG showed limited success for treatment. Due tovarious problems in using BCG, however, the Applicant has sought otheralternative means to activate macrophage and to complement the inventiveThyex compositions.

According to particular aspects, polysaccharides, such as beta glucanconsisting of complex sugars found in cell walls of yeasts andmushrooms, are a preferred agent in combination with the inventive Thyexcompositions, and act synergistically in combating aging and otherrelated conditions.

There are three forms of beta glucan based on the linkages of thecomplex sugars, and these are recognized as beta-1,3 or 1,4, and 1,6glucan. Most are in the form of 1,3 and 1,4, or 1,3 and 1,6, but the 1,3form, which is most abundant in the fruiting bodies of certain mushrooms(e.g., Sparassis crupa or Cauliflower mushroom; or Lentinula edodes orshitake, etc.). Typically, marketing strategies relating to marketingglucan (typically from the cell wall of the common yeast) emphasize“enhanced the immune system,” “increases antibody production,” and“fight cancer.”

A reference by Ohno, Miura, Nakajima, and Yadomae (2000, Biol. Phar.Bull. 23, 866-872) describes a procedure for extracting beta glucan fromshitake mushroom. Recently, two firms in Japan have successfullycultured the cauliflower mushroom (aka Hanabaritake), and the Applicanthas obtained cauliflower mushroom powder form from these firms.

According to particular aspects, a preferred polysaccharide comprisesone or more of the beta glucans, including three types based on thelinkages: 1-3, 1-4, and 1-6). A number of commercial beta glucanproducts are available with most being derived from the common yeast.According to particular aspects, however, the preferred sources aremushrooms; with shitake being most common because of its readyavailability/source, and cauliflower mushroom (Sparassis crupa), whichis preferred as it contains beta 1-3 glucans, but unfortunately haslimited availability. Additionally, the shitake mushroom, which is mostwidely available, is reported to contain the 1-3 glucan and chitin.

According to particular aspects, an oral route of administration isfavorable, possibly because the intestinal walls are sites containinglarge amounts of lymph nodes and thus T cells.

Additional Combination Agents and/or Therapies:

As indicated above, preferred aspects comprise treatment of aging usingThyex compositions in combination with other fungal and/or herbalpreparations, including the following:

Paresis crepe (aka cauliflower mushroom or hanabaritake) preparations,comprising beta 1-3 glucan, can be used to stimulate macrophage incombination with the inventive Thyex compositions.

Lentinula edodes (shitake; e.g., alkaline digest according to theprocedure reported by Ohno et al. (Biol. Phar. Bull. 23 866-872, 2000),comprises beta 1-3 glucan and chitin, and can be used for treatingage-related illness in combination with the inventive Thyexcompositions.

Astralagas membranaceus (Scutellaria baicalensis, Houttuynia cordata;hot water extract of ground herbs and secondary extraction by alkalinedigest as above), stimulate macrophages, and can be used for treatingage-related illness in combination with the inventive Thyexcompositions.

Lilium longiforum (aka Easter lily; to prepare extract, leaves arepre-frozen, blended (homogenized) in water, and boiled. The liquidextract centrifuged and the supernatant solution distilled(approximately one-half volume is collected)), can be used for treatingage-related illness in combination with the inventive Thyexcompositions.

Houttuynia cordata (as mentioned above) extracts from leaves (e.g.,processed as described herein in Example 10) can be used for treatingage-related illness in combination with the inventive Thyexcompositions. According to particular aspects, DYXC has substantialutility to treat nausea in both humans and animals caused by illnesses,infections, or other treatments, and in particular embodiments is usedin combination with one or more of the inventive Thyex compositions,plus or minus standard additional drugs.

Example 10 Houttuynia cordata Extracts

With reference to FIG. 5, this example provides two process embodimentsused to prepare Houttuynia cordata extracts, and compositions (“D-YXC-1,and 2”) produced in accordance therewith.

Houttuynia cordata Thunb, of the family Saururaceae, is a widely knownherb (Houttuyniae) from ancient times, and its medicinal effects(particularly of the essential oils of the aerial parts thereof) havebeen described in various publications relating to herbal medicines(see, e.g., Huang, The Pharmacology of Chinese Herbs, CRC Press, 1999).The steam distillate prepared from fresh plants of Houttuynia cordataThunb has been reported to have in vitro inhibitory activity againstsome, but not all, enveloped viruses (Hayashi et al., Planta Med.61:237-241, 1995). The herb has also been reported to exhibitantibacterial activity (Huang, supra; Hu, Zentralbl. Veterinarmed.44:365-70, 1997). The herb has been used as a tea for many years, but,as recognized in the art, the process of making the tea (e.g., grindingand boiling the Houttuynia cordata) produces a very bitter andunpleasant taste. There are many art recognized methods that attempt toreduce the bitterness of this aqueous extract of Houttuynia cordata,including organic extraction, roasting the herb, or bleach. Many groupshave reported using pressurized organic solvent extraction to reduce thebitterness of hops.

According to particular aspects and as described in this Example, toreduce the bitterness and increase the palatability of the Houttuyniacordata extract, the Houttuynia cordata extract is subjected to furtherseparation using centrifugation and heat-distillation. According tofurther aspects, it is this last step of heat-distillation that removesthe majority of the bitterness and unpleasant taste associated with theHouttuynia cordata extract, and modifies it to a palatable extractsuitable for oral administration. According to yet further aspects, thisheat-distillation process not only provides for separation of theunpalatable and palatable portions, but also allows for separation ofthe anti-nausea and/or anti-emetic activity from the largely unpalatableportion. According to still further aspects, this heat-distillationprocess purifies and concentrates the anti-nausea and/or anti-emeticactivity. According to certain aspects, the separation and/or removal ofthe bitterness from the aqueous extract and the separated aqueousextract using heat-distillation can be separation and/or can be a lossof the bitter flavor.

The commercially-available herb Houttuynia cordata Thunb was eithergrown locally or purchased from a Chinese herb shop (e.g., Star Import,Honolulu) for use in the following embodiments:

D-YXC-1 Process:

The following steps (1)-(7) comprise a process embodiment for producingD-YXC-1, suitable for oral delivery and in particular embodiments, forinhalation:

(1) Aqueous Extraction.

Fresh Houttuynia cordata or previously frozen Houttuynia cordata wasimmersed in a container of boiling water (454 g dried herb/5 L H₂O) andbriefly stirred to disperse the herb. The container was immediatelyremoved from the heat source, covered with a lid and the contentsallowed to “steep” for about 8-10 hours (e.g., overnight) to produce anaqueous extract;

(2) Crude Filtration.

The “aqueous extract” of step (1) was decanted from its container andgravity filtered through one or more layers of standard cheese cloth toproduce a filtered aqueous extract. The steeped herb was compressed(e.g., by hand or mechanical means) to remove as much liquid as possiblefor filtration;

(3) Low-Speed Centrifugation.

The “filtered aqueous extract” of step (2) was centrifuged (this is apreferred step to remove larger cellular debris, which facilitatessubsequent steps) at 3,500×G for 10 minutes at ambient temperature toproduce a pellet, and an aqueous supernatant fraction;

(4) Second Crude Filtration.

The “aqueous supernatant fraction” of step (3) was decanted from thecentrifugation tubes and gravity filtered through one or more layers ofstandard cheese cloth to produce a filtered aqueous supernatantfraction;

(5) Distillation.

The “filtered aqueous supernatant” fraction from step (4) wastransferred to a standard distillation apparatus equipped with atemperature-controlled heating jacket (set at a temperature of slightlygreater than about 100° C.) and a water-cooled condensation arm;Distillation was allowed to proceed until the volume of distillate wasabout half (i.e., about 2 L) that of the initial “filtered aqueoussupernatant” volume to produce a distillate fraction (100 mL increments(from a 1000 mL sample) were tested for the presence of the bitter taste(described herein); after approximately 600 mL the bitter taste wasdetected no longer);

(6) Adjustment of pH and Ionic Strength.

Phosphate buffer (of about pH 7) was added to the “distillate” fractionof step (5) to a final concentration of about 1 mM (e.g., by adding 1 mlof 1M phosphate buffer per liter of “distillate”). Solid sodium chloridewas then added to a final concentration of about 0.85% (wt./volume)(e.g., to a concentration corresponding to “standard physiologicalsaline”) to produce a pH— and ionic strength-adjusted distillatefraction, D-YXC-1;

(7) Filter Sterilization.

The “D-YXC-1” fraction of step (6) was filter sterilized by passagethrough a 0.2μ membrane filter (Millipore) to produce sterile D-YXC-1,suitable for oral delivery or delivery by injection or inhalation; and

(8) Storage.

D-YXC-1, produced in accordance with steps (1)-(7) of the D-YXC-1process, retained stable therapeutic activity when stored either atambient temperature or refrigerated (e.g., 4° C.) in sterilizedcontainers. In particular aspects, D-YXC-1 was dried or lyophilized,stored at ambient temperature and reconstituted (e.g., with sterilewater or other suitable vehicle) prior to use.

D-YXC-2 Process:

The following steps (1)-(2) comprise a process embodiment for producingD-YXC-2, suitable for oral delivery, or for delivery by inhalation:

(1) Preparation of a Distillate Fraction.

Steps (1)-(5) of the above-identified D-YXC-1 process were followed toproduce a distillate fraction, D-YXC-2;

(2) Filter Sterilization.

The “D-YXC-2” fraction of step (1) was filter sterilized according tostep (7) of the above-identified D-YXC-1 process to produce sterileD-YXC-2 suitable for oral delivery or delivery by inhalation (i.e.,aerosol); and

(3) Storage.

D-YXC-2, produced in accordance with steps (1)-(2) of the D-YXC-2process retained stable therapeutic activity when stored either atambient temperature or refrigerated (e.g., 4° C.) in sterilizedcontainers. In particular aspects, D-YXC-2 was dried or lyophilized,stored at ambient temperature and reconstituted (e.g., with sterilewater or other suitable vehicle) prior to use.

General Considerations for Practice of the Above-Identified D-YXC-1 and2 Process Embodiments:

The above-identified steps comprising embodiments of the D-YXC-1 (steps1-7) and D-YXC-2 (steps 1-2) processes may be practiced with variousmodifications, including but not limited to those outlined below, thatare within the scope of the present invention, and with alternatives orsubstitutions that will be recognized by those of ordinary skill in theart as being equivalent to those used herein to produce embodiments ofD-YXC-1 and D-YXC-2.

The D-YXC-1 and D-YXC-2 embodiments of the present invention compriseaqueous extraction steps. Variations in the precise temperature andduration of the aqueous extraction steps are encompassed by the presentinvention. The fresh herb can optionally be cut-up or ground (e.g.,blended in a commercial blender or grinder) to increase the extractablesurface area. Additionally, the fresh herb can be frozen and thawedprior to blending to optimize the extraction process. Preferably, aratio of about 250 gm dried plant tissue to about 1 L water is used, butthe ratio is not critical and the amount of plant tissue may vary fromabout 100 to about 300 gm/L.

The above-described D-YXC-1 and D-YXC-2 embodiments comprisedistillation steps. Variations in the nature of the heat source (e.g.,temperature-controlled heating jacket, or steam distillation apparatus)or the precise temperature of heat source (within a range from about100° C. to 102° C., where 100° C. represents the boiling point of waterat sea level) and duration of the distillation steps will vary accordingto the precise distillation temperature and device used, and are withinthe scope of the present invention. Preferably, distillation iscontrolled by heating the solution at a temperature(s) within a rangefrom 80° C. to 120° C. More preferably, distillation is controlled byheating the solution at a temperature(s) within a range from 90° C. to110° C. Even more preferably, distillation is controlled by heating thesolution at a temperature(s) within a range from 95° C. to 105° C. Mostpreferably, distillation is controlled by heating the solution at thelowest possible temperature that will still permit the solution to boil.

The above-described D-YXC-1 and D-YXC-2 embodiments comprise filtrationsteps. Variation in the mode of filtration or associated manipulationsare within the scope of the present invention. For example, the “aqueousextract” or “aqueous supernatant fractions” corresponding to steps (1)and (3), respectively, of the D-YXC-1 process can optionally be frozento induce precipitation (e.g., of unwanted starchy material) prior tothe corresponding filtration and/or centrifugation steps (2), (3) and(4). Optionally, fresh Houttuynia cordata plants can be frozen for anylength of time prior to processing. Optionally, this pre-freezing offresh plants prior to processing (pre-boiling) assists in breaking andweakening of cell membranes and/or subsequent separation of materials(e.g., starch).

Preferably, the DYXC-1 and 2 distillate compositions are standardized(spectrophotometrically) at a value of about 200 mg (dried herb wtequivalents)/ml. Dosages are discussed herein above under “DoseDeterminations.”

In particular aspects, D-YXC-1 and 2 were dried or lyophilized, storedat ambient temperature and reconstituted (e.g., with sterile water orother suitable vehicle) prior to use.

According to particular aspects, the active anti-emetic factor containedwithin the extract is a very small molecule having a molecular weight ofless than 1,000 daltons, which is adsorbed rapidly and can block thevagus nerve from receiving stimuli due to pain, motion, infection, or asa complication attributed to certain medications (e.g., chemotherapymedications). According to additional aspects, treating nausea withHouttuynia cordata extract results in blockage of these stimuli and thuswithout further stimulation, the vomiting center in the region of themedulla oblongata is sedated.

According to further aspects, DYXC has substantial utility to treatnausea in both humans and animals caused by illnesses, infections, orother treatments, and in particular embodiments is used in combinationwith one or more of the inventive Thyex compositions, plus or minusstandard chemotherapy drugs, for the treatment of cancer.

Example 11 Treatment of Allergy and Autoimmune Disorders Using theInventive Thyex Compositions

In further aspects, the inventive Thyex compositions are used to treatindividuals with allergy and autoimmune disorders (lichen sclerosis setatrophicus, rheumatoid arthritis, psoriasis, progressive systematicscleroderma, lupus, and juvenile diabetes).

Without being bound by theory, the mechanism may comprise stimulation ofsuppressor T cells, which direct B cells producing the allergyantibodies to stop continued activity and control of reactive T cells.

In additional aspects, Thyex has utility for treating (or preventingfurther deterioration of affected tissues/organs) in ALS, celiacdisease, etc.

Example 12 Post-Surgical Treatment and Wound Healing with ThyexCompostions

In further aspects, the inventive Thyex compositions are used inpost-surgical treatment, and/or for improved wound healing.

Applicants have shown that animals in stress/shock will recovery withinless than 30 minutes following injection with Thyex. Trials indicatethat one undergoing surgery will recover much sooner if Thyex isadministered the day of surgery and again at least one day later.

Without being bound by theory, the mechanism may comprise release ofadrenalin from adrenal cortex. Furthermore, trials involvingadministering thyex on wounds have shown that wound healing wasaccelerated, and trials at a plastic surgery facility are underway tocharacterize the full benefits through the application of a Thyexointment on the surgical wounds.

Example 13 Treatment of Emphysema with Thyex Compositions

In further aspects, the inventive Thyex compositions are used in theTreatment of emphysema.

In further aspects of the present invention, ten patients in variousstages of emphysema, but all requiring an oxygen support system, havegradually increased their respiratory capacity measurements throughThyex administration as an aerosol, with a reduction but not eliminationof their dependence on oxygen support systems.

Without being bound by theory, the mechanism may comprise reduction ofinflammation. If so, the accelerated wound healing described above maybe due, at least partially, on this process.

Example 14 Treatment of Swine with Thyex-1, 2 or 3 Compositions wasAffective Against PRRS, and PRRS-Related Reproductive, Respiratory andGrowth Disorders, Including Wasting Syndrome

This example provides in vivo experiments and assays showing thattreatment of PRRSV-infected animals with Thyex-1, 2 or 3, orcombinations of Thyex-1, 2 or 3 with antimicrobial agents (e.g.,antibiotics, or D-YXC-1 or 2) was affective against reproductive, growthand respiratory symptoms of PRRS. Specifically, Thyex- (or Thyex plusD-YXC-) treated gilts and sows returned to estrus more quickly andsubsequently became pregnant more frequently than did non-treatedcontrol animals. Additionally, Thyex- (or Thyex plus D-YXC-) treatedsuckling and weaned pigs matured more rapidly, and spent less time withsecondary conditions and infections (e.g., diarrhea and/or pneumonia)than did non-treated control animals (which either died or manifestedcharacteristic ‘wasting’ syndrome).

Methods and Materials

Swine and Disease Status.

Study group animals were obtained from a single farm swine herd (“testherd”) that was clinically diagnosed with an endemic PRRSV infection.The PRRSV-positive status of the herd was confirmed by standardserological assays (IFA and c-ELISA) performed by the Animal DiseaseDiagnostic Laboratory, Washington State University, Pullman, Wash.Secondary symptoms displayed within the test herd includedbroncho-pneumonia, Salmonellosis, and diarrhea (especially among postweanlings).

Treatment Groups, and Data Presentation.

PRRSV-positive animals from the test herd were divided into “Thyex-1”treatment groups, “Thyex-1 plus antimicrobial agent (e.g., antibioticsor D-YXC-1)” combination treatment groups, and “control” groups thatreceived neither Thyex-1 nor antimicrobial agent. An additional“reference” group consisting of corresponding uninfected swine was usedto normalize data for presentation. Data are reported as a percentage ofthe corresponding uninfected reference group data.

Dosage Regimens.

Animals in the Thyex-1 treatment group were treated with either a three-or up to about a seven-day regimen consisting of daily injections,delivered either intramuscularly (IM) or subcutaneously (SQ), of 1 mlThyex-1 composition (e.g., about 0.2 mg/kg/day to about 0.3 mg/kg/day).Animals in the Thyex-1 plus antimicrobial agent (e.g., antibiotics orD-YXC-1) combination therapy group received the same Thyex-injectionregimens with the addition daily administration of antimicrobial agent(e.g., antibiotics or D-YXC-1). In the case of D-YXC-1 combinationtherapy, twice-daily injections, either IM or SQ, of D-YXC-1 (about 20mg/kg/day to about 25 mg/kg/day), was administered. Antibiotic dosageswere those of the label, according to the particular antibiotic used.

Results

Reproductive Study.

Pregnant gilts and sows from a PRRSV-infected swine herd were treatedwith either Thyex-1 or Thyex-1 plus antimicrobial agent (e.g.,antibiotics or D-YXC-1), or were left untreated for controls. Afterfarrowing and weaning, reproductive data (in the form of % of animalsreturning to estrus relative to an uninfected reference group, and inthe form of % of animals pregnant relative to an uninfected referencegroup) was assessed for all three groups.

The data showed that post-farrowing gilts or sows treated with Thyex-1,or with a combination of Thyex-1 and antimicrobial agent (e.g.,antibiotics or D-YXC-1), showed a significantly more rapid return toestrus and a higher pregnancy rate relative to non-treated,PRRSV-infected control animals. Additional studies showed that theThyex-1 and 2, and the Thyex plus antimicrobial agent (e.g., D-YXC-1 and2 compositions) were substantially equivalent and effective for treatingthese PRRS reproductive symptoms. Thyex-3 treatment was also found to beeffective in this regard, but involved oral administration (about 15ml/day) of the composition over longer treatment periods (e.g., 20-30consecutive days). Preferably, for oral administration, the thyex-3protein concentration was standardized at a protein concentration ofabout 2 mg/ml.

Maturation and Respiratory Study.

Suckling and weanling swine from a PRRSV-infected swine herd weretreated with either Thyex-1 or Thyex-1 plus antimicrobial agent (e.g.,antibiotic or D-YXC-1 and 2 compositions), or were left untreated ascontrols. The animals were placed on feed, and maturation data (in theform of ‘average daily gain’ (ADG) or ‘feed to gain’ (FG) relative to anuninfected reference group), and respiratory data (in the form of theaverage time spent with respiratory symptoms relative to an uninfectedreference group) was assessed for all three groups.

The data showed that weanling swine treated with Thyex-1, or with acombination of Thyex-1 and antimicrobial agent (e.g., D-YXC-1 and 2compositions) matured more rapidly, and spent less time with secondaryconditions and infections (e.g., pneumonia, intestinal disorders, anddiarrhea) than did non-treated, PRRSV-infected control animals. Thenon-treated animals that survived the infection remained as runts (i.e.,PRRSV-related “wasting” syndrome), whereas treated littermates continuedto develop as those in the uninfected control animals. Thus, theinventive treatment was also found to be effective for treatment ofwasting syndrome. As in the case of the reproductive studies, theThyex-1 and 2, and the Thyex plus antimicrobial agent (e.g.,antibiotics, or D-YXC-1 and 2) compositions were found to besubstantially equivalent and effective for treating PRRSV-relatedmaturation and respiratory symptoms, including diarrhea.

Thyex-3 treatment was also found to be effective in this regard, butinvolved oral administration (about 15 ml/day) of the composition toweanlings over longer treatment periods (e.g., 20-30 consecutive days).Preferably, for oral administration, the thyex-3 protein concentrationwas standardized at a protein concentration of about 2 mg/ml.

Example 15 Thyex Compositions are Useful as Adjunct Methods forVaccination Regimens in Swine

Recent outbreaks of atypical or acute PRRS in vaccinated swine raiseserious concern about the efficacy of current vaccines and provideimpetus for developing more effective vaccines and/or other adjunctmethods. For example, the current monovalent (based on a single PRRSVstrain), bivalent or even polyvalent vaccines (based on 2 or 3 strains;see, e.g., U.S. Pat. No. 5,976,537) are not effective in protectingagainst infections of genetically diverse field strains of PRRSV thatroutinely infect “vaccinated” herds (Meng, Vet. Microbiol. 74:309-29,2000). That is, strains are often poorly cross-protective. Moreover,“modified-live” vaccines cause large losses in gestating sows, so thattheir use is, at best, restricted to non-gestating sows (Dewey et al.,Prev. Vet. Med. 40:233-241, 1999; Mengeling et al., Am J. Vet. Res.60:796-801, 1999). Furthermore, substantially attenuated vaccines, whilereducing the occurrence of vaccine-induced reproductive failure whenpregnant sows are vaccinated, are likely to be less cross-protectivethan less attenuated versions.

Finally, PRRSV vaccine approaches, like eradication protocols, do notaddress the practical and financial realities associated withsubstantial swine groups that are already infected with diverse fieldstrains of PRRSV, or with substantial vaccinated swine groups that willnonetheless still become infected with one or more diverse field strainsof PRRSV.

Therefore, according to particular embodiments of the present invention,Thyex-1, 2 or 3, or combinations of Thyex-1, 2 or 3 with antimicrobialagents (e.g., antibiotics, or D-YXC-1 or 2) are useful as an adjuncttherapy or method to enhance the efficacy of PRRSV vaccine elements andvaccination regimens. Specifically, Thyex-1, 2 or 3 alone, or incombination with antimicrobial agents is used as an adjunct treatment tosignificantly enhance the efficacy of vaccination of gilts and sows withvarious PRRSV vaccines.

Accordingly, a PRRSV vaccine (e.g., and attenuated PRRSV vaccine) isused to vaccinate uninfected gilts, in combination with the inventiveadjunct Thyex treatment (e.g., with Thyex-1, 2 or 3, alone or incombination with antimicrobial agents, as disclosed herein). Such PRRSVvaccines are derived, for example, from PRRSV strains NADC-8, NADC-9,and NVSL-14, which normally cause reproductive failure in pregnantgilts. The vaccines are formulated in an effective immunization dosagewith a pharmaceutically acceptable carrier or diluent, such asphysiological saline or tissue culture medium. The “effectiveimmunization dosage” is that amount needed to induce immunity in a pigagainst challenge by a virulent strain of PRRSV; typically 10⁴ to 10⁶median cell culture infectious units (CCID₅₀) (virus is propagated in,e.g., primary alveolar macrophages, or in more generally availableAfrican green monkey kidney cell lines, such as MARC-145 cells or theMA-104 clone thereof, that are available at diagnostic laboratoriesnationally). Preferably, the vaccines are administered oronasally or byinjection. Preferably, appropriate adjuvants may be included in thevaccine formulation. The vaccines are used individually, or arepreferably combined together in the formulation of polyvalent vaccines.

In particular embodiments, gilts are vaccinated either within the firstfew weeks after birth, or at least 2 months prior to mating. The vaccineis preferably administered to gilts at least two months prior to matingto preclude vaccine-induced reproductive failure. Preferably, all pigsare vaccinated within the first few weeks after birth in order toprotect against the respiratory symptoms of the disease. Animals aretreated at or near the time of vaccination, for example, with either atwo- or three-day injection regimen consisting of daily injections of 1ml of Thyex-1 or 2 composition, delivered either intramuscularly (IM) orsubcutaneously (SQ). Alternatively, animals are treated at or near thetime of vaccination, for example, with either a two- or three-dayinjection regimen consisting of daily injections of 1 ml of Thyex-1 or 2composition, delivered either intramuscularly (IM) or subcutaneously(SQ) in combination with the addition of twice-daily injections, eitherIM or SQ, of D-YXC-1 (5 ml/animal). Preferably, Thyex adjunct treatmentoccurs shortly before or at the time of vaccination.

Accordingly, adjunctive therapy with either Thyex-1, 2 or 3 compositions(or with Thyex-1, 2 or 3 plus antimicrobial agent) results in enhancedefficacy of PRRSV vaccination regimens, where the reproductiveperformance of vaccinated sows receiving the adjunct Thyex treatmentexceeds that of corresponding vaccination only control groups. Moreover,fewer live-born pigs from vaccinated sows receiving Thyex adjuncttherapy are infected at birth, compared to the live-born pigs from sowsreceiving vaccination only (no Thyex adjunct treatment).

According to the present invention, adjunct treatment of swine withThyex (or Thyex plus antimicrobial agent) compositions enhances theefficacy of vaccine regimens in protecting PRRSV-susceptiblereproductive systems against virulent field strains of PRRSV (includingstrains NADC-8, NADC-9, and NVSL-14), found in widely differentgeographical areas including Canada, Guatemala, and various states ofthe United States (Wesley et al., Proc. Am. Assoc. Swine Pract. 141-143,1996; and see U.S. Pat. No. 5,976,537).

According to the present invention, sows that are vaccinated incombination with administration of the inventive adjunct treatment showenhanced protective immunity to PRRSV, even during late gestation (e.g.,at about Day 90 of gestation), the time of greatest reproductivesusceptibility to PRRSV due to viral crossing of the placenta (Mengelinget al., Am. J. Vet. Res., 57:834-839, 1996).

According to the present invention, adjunctive treatment of swine withThyex (or Thyex plus antimicrobial agent) compositions enhances theefficacy of vaccine regimens in protecting PRRSV-susceptible respiratorysystems against PRRSV and PRRSV-related disease, including that causedby a variety of virulent field strains of PRRSV.

Example 16 Thyex Compositions are Useful as an Adjunct Treatment toReduce or Preclude Vaccine-Induced Reproductive Failure in Swine

Late gestation in swine (e.g., at about Day 90 of gestation) is the timeof greatest reproductive susceptibility to PRRSV (attributed to viralcrossing of the placenta; Mengeling et al., Am. J. Vet. Res.,57:834-839, 1996), and thus corresponds to a period of greateconomic/commercial importance for the swine breeder.

Vaccination of pregnant swine during gestation is particularlyproblematic, because of the art-recognized problem of vaccine-inducedreproductive failure. This problem can be partially addressed by thedevelopment of substantially attenuated virus strains for use in thepreparation of vaccines to be administered during gestation (see, e.g.,U.S. Pat. No. 5,976,537 to Mengling et al, incorporated by referenceherein in its entirety). However, development of such substantiallyattenuated PRRSV strains is expensive and time consuming, and can resultin the production of less efficacious vaccines. Additionally, new PRRSVstrain variants are frequently occurring in the field, and substantiallyattenuated vaccines are less likely to be cross-protective than lessattenuated vaccines. Preferably, less attenuated and more broadlyprotective vaccines could be administered during gestation, if theassociated vaccine-induced reproductive failure could be reduced orprecluded.

According to the present invention, adjunctive treatment of swine withThyex (or Thyex plus antimicrobial agent) compositions allows for theuse of less attenuated more broadly protective vaccines by significantlyreducing vaccine-induced reproductive failure when such vaccines areadministered during gestation, including late gestation.

Therefore, the compositions and treatment methods of the presentinvention were effective for treating PRRS, PRRS-related conditions andsecondary infections (e.g., diarrhea, pneumonitis and/or intestinaldisorders), and wasting syndrome in pregnant gilts and sows, and inswine being fattened for slaughter. Post-farrowing gilts or sows showeda more rapid return to estrus and a higher pregnancy rate. Additionally,treated suckling and weanling swine recovered more rapidly, relative tonon-treated control animals (which either died or manifestedcharacteristic ‘wasting’ syndrome), from PRRS-associated secondaryinfections, such as pneumonia, and wasting syndrome. Treated sucklingsand weanlings, in contrast to non-treated animals, had healthy appetitesand reached full normal maturity within the same time period “on feed”as control animals that were not infected with PRRSV. Compositionsproduced in accordance with the processes of the present invention canbe administered to breeding gilts, sows, boars, sucklings or weanedpiglets. Preferably, the compositions are administered to breedingfemales before mating, or to pregnant animals to help protect the entirepregnancy period. Preferably, the compositions are administered to youngsucklings and to young pigs shortly after weaning to protect their latenursery, grower and finishing stages.

According to particular aspects, adjunct treatment of swine with Thyexcompositions is effective to enhance the efficacy of vaccine regimens inprotecting PRRSV-susceptible reproductive systems against virulent fieldstrains of PRRSV.

According to additional aspects, adjunct treatment of swine with Thyexcompositions is effective to enhance protective immunity to PRRSVinfection, even during late gestation.

According to further aspects, adjunctive treatment of swine with Thyexcompositions is effective to enhance the efficacy of vaccine regimens inprotecting PRRSV-susceptible respiratory systems against PRRSV andPRRSV-related conditions.

According to yet further aspects, adjunctive treatment of swine withThyex compositions is effective to reducing vaccine-induced reproductivefailure when such vaccines are administered during gestation (includinglate gestation), allowing for the use of less attenuated and/or morebroadly protective vaccines.

Example 17 Houttuynia Cordata Extracts Had Substantial Use as anAnti-Nausea/Anti-Emetic Therapeutic in Mammals

Houttuynia cordata extracts (DYXC) were prepared as disclosed in Example10. A canine patient presented with an acute gum lesion due to pyorrhea.In addition, blood samples indicated that the animal had a systemicinfection and had acute dehydration due to frequently vomiting. Atreatment regime of 2 cc of DYXC placed directly into mouth of theanimal every hour was begun. The intense vomiting halted almostimmediately and within three days the canine began to eat and drinknormally. After seven days of treatment the acute gum lesion due topyorrhea healed completely. The anti-nausea properties of the Houttuyniacordata extracts (DYXC) was confirmed through a number of other cases.

Example 18 Houttuynia Cordata Extracts Had Substantial Use as anAnti-Emetic Therapeutic in Humans

Houttuynia cordata extracts were prepared as disclosed in Example 10. Afemale human patient presented with severe nausea due to thechemotherapy treatment from her stomach cancer. The subject reportedthat the nausea was very taxing. About 30 cc of the Houttuynia cordataextract (DYXC) was administered to the subject (orally) as needed fortreating the nausea. The subject reported rapid abatement of nausea.

Likewise, a male human cancer patient being treated with chemotherapypresented with severe nausea. About 30 cc of the Houttuynia cordataextract (DYXC) was administered to the subject (orally) as needed fortreating the nausea. The subject reported rapid and substantialabatement of nausea within a few minutes after administration.

Example 19 Houttuynia Cordata Extracts has Substantial Use asAnti-Nausea/Anti-Emetic Therapeutics Animals (e.g., Vertebrates,Mammals, Etc.)

Houttuynia cordata extracts are prepared, for example, as disclosed inExample 10. According to certain aspects, the heat distilled extracts ofHouttuynia cordata have substantial use in relieving nausea and/orvomiting. This nausea and/or vomiting can be due to any conditionincluding, but not limited to pregnancy, motion sickness,gastrointestinal obstruction, peptic ulcer, drug toxicity, myocardialinfarction, renal failure, and hepatitis. In addition, nausea and/orvomiting can follow the administration of many drugs particularly cancerchemotherapeutic agents. According to certain aspects, subjectspresenting with nausea and/or vomiting find that these symptoms quicklyabate upon treatment with the Houttuynia cordata heat distilled extracts(DYXC).

Example 20 Houttuynia Cordata Extracts are Further Fractionated toIdentify the Anti-Nausea Agent

As shown in Example 10, Houttuynia cordata extracts can be fractionatedinto portions that contain anti-nausea and/or anti-emetic activity.According to certain aspects, the heat distilled fraction (as disclosedin Example 10) can be further fractionated using separation techniqueswell known in the art (e.g., fractional distillation, centrifugation,chromatography, crystallization, electrophoresis, evaporation,extraction, flotation, flocculation, precipitation, and columnchromatography). The further separated fractions can be screened foranti-nausea and/or anti-emetic activity as described herein. In additionand according to particular aspects, the further separated fractions canbe screened for anti-nausea and/or anti-emetic activity using methodswell known in the art (e.g., using test frogs and/or chicks). Forexample, researchers have used frogs and chicks that were induced tovomit by emetic agents to test anti-nausea and/or anti-emetic agents foryears (Khan, R. A., et al., 2005 “Preliminary Screening of Methanol andButanol Extracts of Tamarindus indica for Anti-Emetic Activity,” J.Basic and Applied Sciences, Vol. 1, No. 2; Kawai, T., et al., 1994“Anti-emetic principles of Magnolia obovata and Zingiber officinale.”Planta Med. 60: 17-20; Kinoshita, K., et al., 1996. “Anti-emeticprinciples of Inula linariaefolia flowers and Forsythia suspensefruits”, Phytomedicine 3: 51-58; Tai, T., et al., 1995. “Anti-emeticprinciples of Poria cocos.” Plants Med. 61: 493-590; Akita, Y., et al.,1998. “New assay method for surveying anti-emetic compounds from naturalsources.” Nat Prod Sci 4(2): 72-77; Yang, Y., et al., 1999. “Anti-emeticprinciples of Pogostemon cabin (blanco) benth.” Phytomedicine6(2):89-93; all of which are incorporated herein by reference in theirentireties and particularly for their teachings relating to assays andscreening methods for detecting/characterizing anti-nausea/anti-emeticagents). Given the presently disclosed novel anti-nausea/anti-emeticactivity of the Houttuynia cordata extracts, coupled with the knowledgeand skill in the art with respect to standard fractionation andpurification methods, particular aspects of the invention provide notonly for the heat-distilled Houttuynia cordata extract (e.g., of Example10), but for routine fractionation, concentration, and/or purificationof the anti-nausea/anti-emetic activity to provide for fractionated,concentrated, and/or purified derivative fractions of the heat-distilledHouttuynia cordata extract (e.g., of Example 10).

The invention claimed is:
 1. A heat-denatured, fractionated thymus extract composition, comprising thymus proteins or polypeptides having molecular weights within a range of 3.5 kDa to 30 kDa, including within 5 to 14 kDa, and lacking thymus proteins or polypeptides having molecular weights greater than 30 kDa and less than 3.5 kDa.
 2. The composition of claim 1, wherein the thymus proteins or polypeptides having molecular weights within the range of 3.5 kDa to 30 kDa are heat-stable under conditions of being heated in solution to a temperature of 75° C.
 3. The composition of claim 1, wherein the thymus proteins or polypeptides having molecular weights within the range of 3.5 kDa to 30 kDa precipitate under conditions of exposure in solution to 0.7 gm/ml ammonium sulfate.
 4. The composition of claim 1, wherein the thymus proteins or polypeptides having molecular weights within the range of 3.5 kDa to 30 kDa precipitate under conditions of exposure in solution to 0.5 gm/ml ammonium sulfate.
 5. The composition of claim 1, further comprising a pharmaceutically-acceptable excipient, carrier of diluent, to provide a pharmaceutical thymus extract composition.
 6. A fractionated thymus extract composition prepared by a method, comprising: homogenizing thymus tissue with aqueous homogenization fluid to produce an aqueous thymus homogenate; removing tissue debris from the aqueous thymus homogenate to produce a primary supernatant; heat denaturing the primary supernatant, and clarifying the denatured primary supernatant by use of at least one of low-speed centrifugation or filtration, to produce a clarified supernatant; separating molecules having molecular weights less than 3.5 kDa from the clarified supernatant; and separating molecules having molecular weights greater than about 30 kDa from the clarified supernatant to provide a heat-denatured, fractionated thymus extract composition comprising proteins or polypeptides having molecular weights in the range of 3.5 kDa to 30 kDa.
 7. The composition of claim 6, wherein in the method of preparing, homogenizing thymus tissue comprises homogenizing thymus tissue with hypotonic aqueous homogenization fluid.
 8. The composition of claim 6, wherein the method of preparing comprises further clarifying of the clarified supernatant by high-speed centrifugation to produce a final clarified supernatant fraction.
 9. The composition of claim 6, wherein the method of preparing further comprises sterilizing the fractionated thymus extract composition.
 10. The composition of claim 9, wherein sterilizing is achieved by passing the final clarified supernatant fraction through a membrane filter.
 11. The composition of claim 6, wherein in the method of preparing, the initial ratio of thymus tissue to aqueous homogenization fluid is about 350 g wet weight of thymus tissue to about 0.7 L of homogenization fluid.
 12. The composition of claim 6, wherein in the method of preparing, removing tissue debris from the aqueous thymus homogenate is achieved by a combination of low-speed centrifugation and crude filtration.
 13. The composition of claim 6, wherein in the method of preparing, heat denaturing and clarifying of the primary supernatant is achieved by heat denaturation, followed by low-speed centrifugation and crude filtration to remove particulate matter.
 14. The composition of claim 6, wherein the method of preparing further comprises lyophilization of the fractionated thymus extract composition.
 15. The composition of claim 6, wherein in the method of preparing, no steps involving exogenously added protease digestion, or extraction with organic solvents are used.
 16. The composition of claim 6, further comprising a pharmaceutically-acceptable excipient, carrier of diluent, to provide a pharmaceutical thymus extract composition.
 17. A fractionated thymus extract composition prepared by a method, comprising: homogenizing thymus tissue with aqueous homogenization fluid to produce an aqueous thymus homogenate; removing tissue debris from the aqueous thymus homogenate to produce a primary supernatant; heat denaturing the primary supernatant, and clarifying the denatured primary supernatant by use of at least one of low-speed centrifugation or filtration to produce an intermediate clarified supernatant; concentrating the intermediate clarified supernatant to produce a concentrated intermediate fraction; separating molecules having molecular weights less than 3.5 kDa from the concentrated intermediate fraction; and separating molecules having molecular weights greater than 30 kDa from the concentrated intermediate fraction to provide a heat-denatured, fractionated thymus extract composition comprising proteins or polypeptides having molecular weights in the range of 3.5 kDa to 30 kDa.
 18. The composition of claim 17, wherein in the method of preparing, homogenizing thymus tissue comprises homogenizing thymus tissue with hypotonic aqueous homogenization fluid.
 19. The composition of claim 17, wherein the method of preparing comprises further clarifying of the clarified supernatant by high-speed centrifugation to produce a final clarified supernatant fraction.
 20. The composition of claim 17, wherein the method of preparing further comprises adjusting at least one of the pH or ionic strength of the fractionated thymus extract composition having proteins or polypeptides of molecular weight of about 3.5 kDa to about 30 kDa to a physiological or therapeutically compatible value, to produce a pH-adjusted fraction or ionic strength-adjusted fraction.
 21. The composition of claim 20, wherein adjusting at least one of the pH or ionic strength to a physiological or therapeutically compatible value is achieved by adding phosphate buffer or sodium chloride to produce a fraction having at least one of a pH value of about 7 or an ionic strength of about 0.85% w/v.
 22. The composition of claim 20, wherein the method of preparing further comprises sterilizing the pH-, or ionic strength-adjusted fractionated thymus extract composition to produce a sterile pH-adjusted fraction, or ionic strength-adjusted fraction.
 23. The composition of claim 22, wherein in the method of preparing, sterilizing is achieved by passing the fraction through a membrane filter.
 24. The composition of claim 17, wherein in the method of preparing, the initial ratio of thymus tissue to aqueous homogenization fluid is about 350 g wet weight of thymus tissue to about 0.7 L of homogenization fluid.
 25. The composition of claim 17, wherein in the method of preparing, removing tissue debris from the aqueous thymus homogenate is achieved by a combination of low-speed centrifugation and crude filtration.
 26. The composition of claim 17, wherein in the method of preparing, heat denaturing and clarifying of the primary supernatant is achieved by heat denaturation, followed by low-speed centrifugation and crude filtration to remove particulate matter.
 27. The composition of claim 17, wherein in the method of preparing, concentrating the intermediate supernatant involves concentrating and fractionating, and wherein the concentrating and fractionating is achieved by adding ammonium sulfate to the intermediate clarified supernatant, followed by low-speed centrifugation and suspension of the resulting ammonium sulfate pellet in an aqueous solution to provide a concentrated intermediate fraction.
 28. The composition of claim 17, wherein in the method of preparing, separating molecules having molecular weights less than about 3.5 kDa from the concentrated intermediate fraction comprises dialysis of the concentrated intermediate fraction, followed by high-speed centrifugation to remove particulate matter, to provide for a clarified concentrated intermediate fraction lacking proteins or polypeptides having molecular weights less than about 3.5 kDa.
 29. The composition of claim 28, wherein in the method of preparing, separating molecules having molecular weights greater than about 30 kDa from the heat-treated, fractionated thymus extract composition, is achieved by passing the clarified concentrated intermediate fraction lacking proteins or polypeptides having molecular weights less than about 3.5 kDa consecutively through a first and a second membrane filter having exclusion limits of about 100 kDa and about 30 kDa, respectively, and collecting the filtrate.
 30. The composition of claim 29, wherein the method of preparing further comprises lyophilization of the heat-treated, fractionated thymus extract composition comprising proteins or polypeptides having molecular weights in the range of about 3.5 kDa to about 30 kDa.
 31. The composition of claim 17, further comprising a pharmaceutically-acceptable excipient, carrier of diluent, to provide a pharmaceutical thymus extract composition. 